Labeling, isolation, and characterization of cell type-specific exosomes derived from murine skin tissue

Abstract

Extracellular vesicles (EVs) are a heterogeneous group of membrane-bound vesicles involved in cell communication, formed at the plasma membrane (ectosomes) or by endocytosis (exosomes). Most exosome studies to date have focused on in vitro systems or exosomes derived from body fluids, while tissue-derived exosomes remain underexplored. Here, we present a protocol employing cell type-specific promoter-driven reporter constructs for targeted labeling and subsequent isolation of exosomes from specific cell types in vivo from murine tissues. The differentiation between exosomes and ectosomes remains challenging due to limitations of current isolation techniques that are primarily based on size, density, or surface markers. To address this issue, our approach leverages genetic engineering to mark exosomes specifically, allowing for their precise identification and isolation from a complex biological pool of heterogenous EVs. The isolated cell-specific exosomes are characterized by electron microscopy, nanoparticle tracking analysis, antibody exosome array, and other established techniques. The labeling and isolation of exosomes spans 2–3 days and is designed to be accessible to researchers with fundamental laboratory competencies. This protocol facilitates the study of exosome-mediated communication by enabling the isolation of cell type-specific exosomes from either individual cell types or multiple cell types in combination. Most experiments within the protocol have used murine wound-edge (WE) skin tissue, but the protocol can in principle also be applied to other tissues to isolate exosomes with a few modifications as required. This methodology opens new avenues for exploring the functional roles of cell type-specific exosomes in intercellular communication.

Introduction

Recent findings have established a critical role of extracellular vesicles (EVs), cell-released membranous structures in cell-cell communication.^1–7 The EV research community has witnessed significant growth in recent years, spearheading initiatives to elucidate the biological roles of EVs within human physiology and pathology.^8,9 From 1991 to 2021, global research activity related to EVs has markedly increased, as evident from a sharp increase in the number of scientific publications describing their physiological and pathological functions.¹⁰ The field of EV research is anticipated to sustain its upward trajectory, focusing on developing innovative EV-based diagnostic and therapeutic approaches, which are poised to make significant contributions to human healthcare.^9,11,12 Prospective research areas of interest include, but are not limited to, oncology, musculoskeletal, respiratory, urinary, endocrine, neurological, and cardiovascular systems.^13,14 Despite this progress, several critical scientific challenges remain, including the need for a deeper understanding of EV heterogeneity, mechanisms underlying cargo loading, and the development of precise EV metrology.^15–17

Extracellular vesicles include a diverse set of nano to microscale membranous structures.¹⁸ Majority of EVs (Ø 50–1000 nm) originate from the plasma membrane and are called microvesicles, microparticles, membrane particles, and apoptotic bodies that are collectively termed ectosomes.¹⁸ Thus, the study of EVs is inherently challenging because it encompasses numerous families of vesicles.^19–21 Unlike other membrane-originating nanoscale ectosomes (Ø 50–1000 nm),^18,22 exosomes (Ø 50–150 nm) are vesicles of endocytic origin that selectively package a distinctive repertoire of cargo such as ribonucleic acids (mRNA/miRNAs) containing a distinct EXOmotif (short sequence motifs in miRNAs) and other cargoes such as proteins, lipids, metabolites, and membrane receptors.^23–25 The cells possess distinct sorting mechanisms that guide specific intracellular miRNA to enter exosomes.^26–28 Thus, size-based separation techniques are unsuitable for separating multiple possible identities of similar-sized vesicles, limiting the ability to delineate specific mechanisms.^7,29–31 In addition, because Minimal Information for Studies of Extracellular Vesicles (MISEV) reporting standards are relatively recent, numerous studies do not report enough methodological details necessary to reproduce the findings.^29,32 As sophisticated work on well-characterized exosomes has started to emerge,^24,33–36 it is important to acknowledge that much of the early literature on “exosomes” and related data interpretation is clouded and sometimes misleading because these studies do not adhere to appropriate rigor set by MISEV guidelines.¹⁶,³²,^37–39

The endosomal-originated exosomes with specific molecular signals encoded in the form of glycans, miRNA, lipids, or protein signatures are known to participate in cell-cell crosstalk at the site of tissue repair.^{1–7,40–42} However, current isolation strategies based on size, density, or surface markers fail to discriminate between vesicles of differing biogenic origins, thus hindering accurate classification into exosomes or ectosomes.^43,44 Consequently, as per MISEV 2023 guidelines, the scientific community frequently employs the more encompassing term ‘large or small extracellular vesicles’ to circumvent mislabeling and ambiguity.¹² The prevailing methods for bulk characterization fall short of addressing the heterogeneity within EV preparations, offering merely an average representation of the EV cohort. Therefore, interpretations of data derived from these EV populations must be approached with caution due to their inherent variability. Furthermore, within the tissue, the presence of multiple cell types adds another level of complexity to the process of exosome isolation. The field presently anticipates the development of refined methodologies for the isolation, segregation, and characterization of cell type-specific exosomes. Such advancements would facilitate the establishment of a more precise and functional taxonomy in cell-cell communication biology.

Development and overview of the protocol

A significant challenge in EV research is the heterogeneity and inefficiency of current isolation methods to purify distinct populations, leading to mixed EV samples.^45–47 To circumvent the EV complexity found in vivo in complex tissues, we developed a method of labeling and isolation of cell type-specific exosomes in vivo that overcomes the inherent complexity of EVs in tissues. According to EVPedia⁴⁸ and Exocarta,⁴⁹ the three tetraspanins CD9, CD63, and CD81 are reliable markers for the identification of exosomes.⁵⁰ We designed three plasmids encoding murine keratin 14 (Krt14) promoter-driven CD9, CD63, and CD81 fused with "in frame" GFP reporters^51,52 (Fig. 1a, b, Supplementary Note 1). This approach allows the delivery of cell type-specific promoter-driven plasmids for labeling and isolating cell type-specific exosomes from murine skin tissue in vivo.^53,54 The research articles demonstrating the protocol’s development, published in 2020⁵³ and 2022,⁵⁵ report the topical delivery of promoter (Krt14)-driven tetraspanin-GFP fusion expression constructs via Tissue Nanotransfection (TNT),⁵⁶ followed by isolation of keratinocyte-derived exosomes using reporter (GFP)-magnetic trap beads (Exo_κ-GFP). Here, we present an optimized protocol for exosome isolation followed by detailed characterizations (Fig. 2). This protocol employs a combination of differential centrifugation steps and immunomagnetic separation using anti-GFP magnetic agarose beads, allowing us to isolate a specific subpopulation of EVs i.e., exosomes. The GFP agarose beads facilitate the specific capture of exosomes expressing the GFP-reporter. By employing an electroporation-based strategy at varying field strengths, we can achieve deeper plasmid delivery into the tissue. Specifically, applying 200 V/mm allows plasmid transfection into the dermal layer, which may facilitate the isolation of exosomes from distinct cell types. This method is also suitable for the isolation of cell type-specific exosomes following excisional wounds on dorsal murine skin at different time points to study the cargo composition and surface modifications of exosomes isolated from the wound edge (WE). Additionally, transfection of plasmids bearing Lyz promoter with RFP and Col1a1 promoter with mNeonGreen enables the specific capture of exosomes released by macrophages, and fibroblasts, respectively, thus enhancing our ability to characterize and study cell type-specific exosomal populations⁵⁴.

Fig. 1 |. Plasmid construct for labeling and isolation of keratinocyte-derived exosomes.

a, Plasmid map of murine keratin 14 (Krt14) promoter-driven expression constructs that encode for CD9, CD63, or CD81 with “in frame” GFP-reporter. b, Schematics of the plasmid cocktail containing plasmids 1–3 used for labeling and isolation of Exoκ-GFP. Panels a and b were created with BioRender.com.

Fig. 2 |. Schematic representation of the steps involved in isolating keratinocyte-derived exosomes from tissue.

a, Schematics showing tetraspanins fused with GFP expressed on the exosome membrane. b, Schematic diagram showing the pulverization of tissue (i: the tissue (which appears red) is transferred to the mortar; ii: liquid nitrogen is added to the tissue in the mortar and iii: tissue is crushed to powdered form with help of pestle) and then Exoκ-GFP isolation process from murine skin tissue post-TNT with plasmid cocktail. ρ = 1.12 – 1.19 g/ml refers to the density of exosomes. Scanning electron microscopy (SEM) images demonstrate the presence of Exoκ-GFP on GFP magnetic beads. Scale, 5 μm. The morphology of the isolated exosomes were analyzed by TEM. Scale, 5 μm. This method of exosome isolation was reported in EV-track (EV-track consortium, and received an EV-METRIC score of 100% (EV Track ID: EV220292). Panels a and b were created with BioRender.com.

The development and validation of this protocol proceeded and optimized through a series of carefully controlled and empirically tested steps (explained at later stages in the protocol), ensuring its reliability, sensitivity, and broad applicability for evaluating exosome functionality. The initial priority was to validate the specificity of the reporter system by confirming that reporter gene expression was not observed in murine tissue (specifically in mammals) and that the selected antibodies (like GFP) displayed no nonspecific binding. These stringent assessments minimized background signals and confirmed that all detected signals originated solely from the target exosomes. Subsequently, considerable efforts were focused on refining the antibody-bead conjugation parameters, including antibody concentration, pH, buffers and incubation time, to yield stable and functionally robust complexes capable of reproducibly capturing exosomes for downstream analyses.

With optimized specificity and conjugation conditions, the focus shifted to verifying promoter-driven expression in primary murine cell types. This step was critical for ensuring selective transcriptional activation of the delivery of plasmids, thereby preventing spurious reporter activity and safeguarding the protocol’s accuracy. In tandem, the selection of a suitable reporter gene proved essential. Given the heterogeneity of exosomes and their diverse cellular origins, identifying a reporter that neither overlapped with endogenous proteins nor elicited cross-reactivity was paramount. Tests were conducted across multiple primary cell types to confirm that the selected reporter could be effectively integrated and reliably detected in varied cellular contexts without confounding signals (see Anticipated Results).

Finally, the bead-based isolation method of exosomes underwent rigorous validation via different characterization techniques. Comparative analyses of cell type-specific exosomes isolated from murine skin tissues with and without the reporter were performed. Nanoparticle tracking analysis (NTA) verified that the isolated particles were in the recognized exosomal size range of 30–150 nm, while scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrated the expected vesicular morphology. Exosome antibody array assay and flow cytometry analysis further confirmed the enrichment of established exosomal/endosomal markers, underscoring the specificity and efficacy of the isolation procedure. This systematic approach— encompassing stringent reporter specificity testing, finely tuned antibody-bead conjugation, careful promoter selection, and comprehensive vesicle characterization—produced a robust, reproducible, and broadly applicable protocol. This methodology offers a reliable and standardized framework for detecting, capturing, and analyzing cell type-specific exosomes across diverse biological systems.

The major stages of the procedure are listed in Table 1.

Table 5 |.

Troubleshooting table

Step	Problem	Possible Reason	Solution
Preparation and quality control of TNT device (Experimental design)	Some of the hollow microchannels are blocked or not opened yet.	The etching time is not long enough, or contamination and particles exist in some holes.	Etch until the clogged channels are opened. Keeping the fabrication environment clean and avoiding contamination from particle sources are important.
Mounting of TNT chip (Experimental design)	Leakage and damage of TNT chip.	The thickness of Polydimethysiloxane (PDMS) membrane for mounting is not appropriate.	Damage can be checked by SEM. For leakage, place the TNT device on a Whatman filter paper, fill the reservoir with 0.01% (w/v) bromophenol blue solution, and incubate for 2–5 min. Any leakage will turn the filter paper blue. Hence, remounting is needed to ensure proper sealing between the reservoir and the chip and ensure that the thickness of the PDMS film is within 2–4 mm during fabrication.56
NTA (Step 109)	Machines sometimes become contaminated by air bubbles or impurities.	Presence of air in the syringe needle while pushing the exosomal dispersion into the inlet.	Use the plunger to push out the water droplet, and the syringe with 10 ml of UPW to flush and effectively eliminate the air bubbles.
Plasmid Transformation (Step 12)	Bacterial colony growth was observed on the negative control plates.	Contamination of LB broth or LB agar plate.	Always tighten the lid of the media after autoclaving and keep at 4 °C. Always prepare LBA petri dishes inside the aseptic hood.
Promoter validation (Step 31)	Low gene expression.	Not enough plasmid was delivered, or the plasmid was not transfected deeply enough.	Increase the plasmid concentration or increase the number of pulses or the voltage.
Nairing and TNT on mouse skin (Step 45)	The animal is not breathing properly.	Anesthesia is too high, or air circulation is not proper.	Lower the anesthesia or enhance the airflow rate.
Exosome antibody array assay (Step 154)	Low intensity of signal.	Short incubation and exposure time	2–3 min of incubation in the developer and enhancer mixture should suffice.
Flow cytometry (Step 158)	Beads stuck in the fluidics system.	agglomeration of beads.	Disperse the sample on beads in 500 μl of 0.1% (v/v) PBST. After dispersing the beads, run the samples again, and it is advisable to backflush the instrument after each reading.

Applications of the method

We demonstrated our protocol's efficiency in isolating keratinocyte-derived exosomes from murine skin tissues using various characterization techniques, including size dispersion, concentration, morphology, and the presence of endosomal markers. Our previous publications highlighted the protocol's capability to isolate specific subpopulations of EVs, particularly exosomes, with high efficiency.^53,54 This advancement will help researchers address the complexities arising from EV heterogeneity, which cannot be adequately separated using traditional techniques. The separation and characterization of exosomes is crucial for advancing our understanding of their biological roles in physiological and pathological conditions. Profiling the molecular cargo of exosomes allows the identification of signature genes, proteins, lipids, glycans, and specific signaling pathways associated with disease progression. Our previous publication in Analytical Chemistry measured the mass distributions and surface charge by charge detection mass spectrometry (CDMS) suggesting that Exoκ extracted from day 5 WE tissue from nondiabetic mice were enriched in high mass compared to Exoκ extracted from day 5 WE from their littermate homozygous diabetic mice. This difference may arise from differences in either cargo packaging or release⁵⁵. Such observations may prove insightful while optimizing biological therapeutics. This knowledge underpins the development of potential exosome-based therapies for the treatment of wounds in diabetic animals, for clinical trials, and pharmaceutical production. Additionally, our protocol serves as a reference for optimizing the isolation and characterization of other cell type-specific EV populations. This method of exosome isolation was reported in EV-track (EV-track consortium, which has helped standardize systematic reporting and methodology by assigning a metric score based on reported experimental parameters²⁹) and received an EV-METRIC score of 100% (EV Track ID: EV220292).⁵⁷

Comparison with other methods

The challenge of effectively isolating cell type-specific exosomes of endosomal origin from other membrane-derived vesicles hinders exploring their role in active intercellular communication.⁴³ While numerous methods have been developed for the isolation of EV from cell culture supernatants and bodily fluids,^58–63 protocols specifically targeting the isolation of exosomes from a specific cell type within a tissue are sparse and largely still in development. Here, we compare our protocol against existing and published EV or exosome isolation methods.^45,61,64,65 This comparison emphasizes the type of tissue that can be utilized for our approach, the impact of various treatments on exosome yield and quality, the required time, and the specific techniques employed for optimal exosome isolation.

Ultracentrifugation is widely regarded as the gold standard for exosome isolation,⁶⁶ employed by nearly 80% of researchers in this field. As per MISEV 2023, the use of the generic term EV and operational extensions of this term can be continued with caution if one or more EV subtypes are separated based on characteristics such as size, density, molecular composition, or cellular origin. These separation methods isolate EV populations with overlapping size profiles and may co-isolate or aggregate unwanted non-EV materials.¹² Some of the other major criticisms of ultracentrifugation are low reproducibility, low yield, aggregation, and mechanical damage to exosomes due to high centrifugal force.^67,68

The high-resolution iodixanol density gradient fractionation method to isolate intermediate extracellular vesicles (IEVs) and small extracellular vesicles (sEVs) from plasma pellets was successful in separating plasma-derived EVs from high-density lipoprotein (HDL) particles. However, this approach fails to effectively remove low-density lipoproteins (LDL), very low-density lipoproteins (VLDL), intermediate-density lipoproteins, and chylomicrons, that are present in human plasma.^61,69 Additionally, the potential for co-isolation of non-vesicular extracellular particles (NVEPs), such as lipoproteins, with exomeres and supermeres cannot be ruled out.⁷⁰

While microfluidic-based techniques for EV analysis offer benefits such as speed, high throughput, and reagent efficiency, challenges remain in the standardization of isolation and detection protocols.⁷¹ Microfluidic-based exosome isolation methods fundamentally rely on precisely engineered channels and controlled flow conditions to separate EVs based on physical parameters such as size, affinity, or other biophysical traits. While this principle works efficiently in well-characterized, relatively homogenous samples, it struggles to capture cell type-specific exosomes from biological fluids or tissue homogenate. Complex tissue and body fluids contain exosomes from multiple cell types with varying surface markers, and cargo profiles.⁷² This heterogeneity can outpace the simplistic sorting criteria that microfluidic platforms employ.⁷³ As a result, these systems fail to yield cell type-specific exosomes that inform disease mechanisms, biomarker discovery, or therapeutic efficacy. Ultimately, although microfluidics can enhance isolation purity under controlled in vitro conditions, its reliance on predefined sorting principles does not adequately address the rich biological variability inherent in physiologically relevant samples.^72–74 Variability in sample preparation, device design, and operating conditions are some of the significant drawbacks that can lead to inconsistent results, compromising the reproducibility and reliability of findings. These issues highlight the need for more robust, standardized methods to advance EV research.

We have rigorously compared exosome isolation via differential ultracentrifugation followed by immunomagnetic separation and only immunomagnetic separation.⁵³ No significant differences in size, shape, or binding properties were observed between exosomes isolated using these two methodologies.⁵³ When isolating exosomes from tissues, it is critical to retain exosomal membrane integrity to maintain the functional properties of exosomes and reduce contamination risk. For instance, using a tissue lyser for EV isolation, as done in some studies, may damage membrane integrity and increase contamination from intracellular vesicles.^75–77 Techniques such as vortexing at later stages of isolation also pose risks. To mitigate these issues, we have avoided mechanical approaches that may damage the membranes during the latter stages of exosome isolation, ensuring exosomal integrity and functionality preservation, and the same was demonstrated by using characterization techniques such as NTA and electron microscopy. We have also checked the purity of the sample by performing immunoblotting of GM130 and Prohibitin, which are reported as major contaminants of exosome preparation by EV-track in our previously published paper. The band of GM130 in the exosome antibody array assay may be possibly due to injury response.⁵³

A major challenge in the initial stages of exosome isolation is the effective release of exosomes from the extracellular matrix. Due to the complex nature of the starting material, the initial tissue treatment is crucial for obtaining adequate quantities of exosomes.^43,78 The enzymatic digestion methods for exosome release from the tissue may compromise the integrity of exosomal surface proteins and overall functionality. The protocol involves pulverizing tissue in liquid nitrogen as fine powder and then incorporating the addition of phosphate-buffered saline (PBS) followed by thorough vortexing to facilitate the release of exosomes from tissues. Once we have the content in PBS, we avoid vortexing at later stages. Subsequent vortexing is avoided to maintain the integrity of exosomal membranes and preserve their functionality. Our characterization methods have confirmed that this process does not compromise the properties of the exosomes, thereby validating the protocol for isolating exosomes from in vivo tissues across various lineages based on the promoter specificity of that lineage.⁵³

Studies previously reported that tissues were cultured ex vivo for extended periods to release EVs or exosomes.^79,80 While this can be a valid approach, the release of exosomes depends on the microenvironmental cues. Thus, exosomes isolated following such ex vivo procedures might differ from those directly isolated from freshly collected tissues. Many researchers have focused on isolating a single EV population, often mislabeling multiple EV subpopulations as one due to overlapping sizes.^45,69,81,82 Other research groups have comprehensively analyzed isolated EV populations using relatively time-efficient methods.^45,83 However, these approaches generally do not focus on isolating distinct EV subpopulations, which may limit their ability to uncover EV subsets' specific characteristics and functions.

In conclusion, our protocol:

1. can be used for the isolation of cell type-specific endosomal-originated exosomes from murine skin tissue in vivo (although with appropriate modifications, it could be applied to other tissues as well).

2. uses a combinatorial approach of differential centrifugation and antibody-conjugated bead based pulldown for the specific isolation of keratinocyte-derived exosomes.

3. minimizes impact on membrane integrity and reduces contamination.

By combining these techniques, our protocol offers a robust and versatile approach for exosome isolation, ensuring high purity and specificity.

Expertise needed to implement the protocol

First-hand isolation of exosomes necessitates experience in basic molecular biology techniques such as transformation and plasmid purifications, performing TNT (or any other topical plasmid delivery approach) for in vivo plasmid delivery, handling mice and performing humane animal euthanasia, and rigor in tissue collection to ensure the reproducibility of data. Since the protocol involves biological samples from mice, adequate precautions must be taken. The Institutional Animal Care and Use Committee (IACUC) must approve animal based research, testing, and teaching before beginning the work. The Institutional Biosafety Committee (IBC) oversees and approves all research programs involving biological materials, including recombinant DNA materials. The isolated exosome populations, obtained through differential centrifugation followed by immunomagnetic precipitation using GFP-agarose beads, should be characterized using several techniques such as NTA, TEM SEM, flow cytometry, and exosome antibody array assay. Techniques like NTA and flow cytometry require specialized laboratory training, while TEM and SEM typically necessitate access to specialized core facilities.

Limitations

The tissue-derived cell type-specific exosome isolation protocol presented here requires successful plasmid delivery. As long as the plasmid delivery system is effective and the selected promoter drives the reporter expression specifically within the target cell type, isolating cell type–specific exosomes becomes highly feasible. By ensuring that only the cells of interest express the fluorescent or otherwise distinguishable reporter, the resulting exosomal population will predominantly originate from that particular cell type. This strategy helps circumvent the challenges posed by cellular heterogeneity in complex tissues, enabling the collection of purer and more functionally relevant exosomes without significant cross-contamination from neighboring cells. However, the TNT technique for plasmid delivery has inherent limitations, including potential tissue damage and the necessity for specialized equipment and optimized electrical parameters based on targeted tissue types.⁵⁶

This methodology, in principle, is applicable for studying exosomes derived from any cellular origin in vivo, leveraging various transgenic murine models to address specific scientific questions. However, cell type-specific isolation of exosomes from human clinical samples, although feasible as reported by us⁸⁴, does not involve using plasmids encoding promoter-driven expression constructs and TNT. While the isolation of cell type-specific exosomes represents an exciting frontier for potential therapeutic applications, the current technology and methodologies remain insufficient for clinical translation. Despite significant progress in preclinical research, several critical barriers highlight the need for further development before cell type-specific exosome labeling and isolations can be safely and effectively employed in clinical settings.

A key challenge for clinical translation is ensuring that cell type-specific exosome labeling methods are biocompatible and do not introduce toxicity. Most existing labeling strategies rely on synthetic dyes, fluorescent reporter proteins, or engineered materials that, while effective in experimental settings, pose potential risks in vivo. These agents may:

• elicit immune responses or inflammation when introduced into the human body.

• alter the native biological properties of exosomes, such as their surface chemistry, cargo composition, or cellular uptake mechanisms.

• accumulate in non-target tissues, raising concerns about long-term safety and potential off-target effects.

For clinical translation, labeling agents must meet stringent safety standards, including being biodegradable, biocompatible, and inert once metabolized. Moreover, the labeling process must preserve the exosomes’ intrinsic functionality, ensuring that their therapeutic efficacy or natural role in cell-cell communication remains unaffected.

Another critical barrier to clinical translation is the lack of standardized protocols for exosome labeling and isolation. Differences in techniques can significantly impact the purity, yield, and functionality of isolated exosomes, leading to variability in experimental outcomes. This inconsistency poses a significant risk when scaling these approaches for clinical applications, where reliability and reproducibility are paramount. Systematic studies that compare labeling methods and their impact on exosome properties are urgently needed.

At present, cell type-specific exosome labeling and isolation methods are still far from being ready for clinical translation. While preclinical research has laid a strong foundation, the field must overcome substantial safety, imaging feasibility, and regulatory compliance challenges. Addressing these gaps through systematic research and innovation will be essential in unlocking the clinical potential of exosome-based therapies. Until then, cell type-specific exosome labeling and isolation will remain a powerful tool for basic research.

Experimental Design

Plasmid design and validation

The designing of the reporter plasmids is a critical step that necessitates thorough validation. Here, we provide an example of labeling and isolating keratinocyte-derived exosomes. The CD9, CD63 or CD81 markers were inserted "in frame" between Lys152 of Krt14 and Met1 of GFP without a stop codon and cloned into the pLenti plasmid backbone. These constructed plasmids were commercially synthesized by ABMGood (www.abmgood.com). The Krt14 promoter-driven plasmids ensure that GFP-tagged CD markers are expressed exclusively in keratinocytes. Given that GFP originates from Aequorea victoria (phylum Cnidaria), there is no endogenous expression in mice, allowing GFP expression to be solely attributed to the activation of the Krt14 promoter (Table 2).

Promoter selection

• Identify the specific cell type of which the exosomes need to be labeled and isolated.

• Select an appropriate tissue-specific and cell type-specific promoter. (https://www.vectorbiolabs.com/promoter-selection-guide/ is a good resource for choosing the right promoter)

Choosing the expression vector

• Select a vector compatible with the mammalian cells. In this work, we choose a pLenti vector. This vector can be packaged in lentivirus for delivery without a topical nanoelectroporation system.

Designing the gene of interest

• Optimize the gene sequence: Adapt the gene sequences of CD9, CD63, and CD81 for optimal expression in mice by performing codon optimisation. This is not necessary when using coding sequences from the same species. The GFP was chosen as a reporter for exosome labeling and isolation from keratinocytes. Different reporters can be used for labeling and isolation of exosomes from multiple cell types as shown in Fig. 3a, b.

Fig. 3 |. Visualization of exosomes originating from different cell types in murine skin tissue.

a, Immunofluorescence image of d7 WE murine tissue showing the presence of Exoκ-GFP from keratinocytes (green), macrophages (red), and fibroblasts (blue) in the dermis. The nuclei are shown in white. Scale, 500 μm (left) and 2 μm (middle and right). b, Imaging mass cytometry⁵⁴ provides better resolution for visualizing the distribution of Exoκ-GFP derived from keratinocytes (green, white arrowhead), and macrophages (red, grey arrowhead). Scale, 200 μm (left) and 2 μm (right).

Optimal plasmid concentrations and buffers:

• Use ~100 ng/μl of each plasmid in PBS for plasmid delivery experiments via TNT.

• Mix the three plasmids in a 1:1:1 ratio with final concentrations ranging from 0.05 to 0.1 μg/μl in PBS, Tris-EDTA, or ultrapure water (UPW) for TNT experiments.

• Plasmids up to ~15 kbp can be delivered using the TNT method.

Specificity testing:

• Test the specificity of the plasmid cocktail in murine keratinocytes, fibroblasts, and macrophages.

• For rigor, test the specificity of the Krt14 promoter in vivo using only the Krt14-GFP plasmid.

This validation method ensures efficient and specific labeling and isolation of cell type-specific exosomes, facilitating detailed studies of exosome biology in various cell types in vivo.

Preparation and quality control of the TNT device.

We have previously reported that the cutaneous delivery of plasmids via TNT (published in Nature Protocols⁵⁶) was efficient and effective. Prior to the delivery of the plasmid cocktail, it is critical to inspect the TNT device for any physical damage and to assess the thickness of the Polydimethylsiloxane (PDMS) utilized to secure the reservoir on the TNT chip. The PDMS film thickness should be precisely adjusted to between 2–4 mm; films thicker than 4 mm can lead to incomplete bonding and potential chip damage, while films thinner than 2 mm may produce wrinkles and air gaps. After mounting, the TNT device is susceptible to damage and leakage⁵⁶ and should thus be checked before the onset of the experiment as described in the Troubleshooting section.

In vivo TNT application.

The efficiency of plasmid delivery via nanoelectroporation is dependent on the applied electric voltage, which influences both the migration force on charged DNA molecules and the delivery distance under a constant pulse duration. Additionally, voltage affects the density and average radius of pores formed on the cell membrane. The electric pulse must be optimized to maintain cell viability and successful delivery of plasmids. An optimal voltage range of 100 V/mm to 200 V/mm balances delivery distance and pore formation parameters. Excessive voltage levels can cause cellular and tissue damage. It is equally important to select the appropriate size of needle arrays. Although the silicon needle shows excellent strength under compression forces, longer needles can be damaged by strong bending or shear force.

This protocol outlines the application of TNT on murine skin via electroporation. For in vivo delivery, male mice aged 8–12 weeks were used, though the protocol applies to any murine strain and is not limited by sex or age. Using an empty vector control is crucial for in vivo experiments.

Preparation of the murine skin:

• Topical TNT-mediated delivery necessitates skin tissue exfoliation to ensure adequate contact between the deeper skin layers and the TNT chip surface. Improper removal of dead cells can lead to blockage of TNT channels, requiring routine cleaning and chip replacement.

Electroporation:

• A proper orientation of the chip onto the skin is a must, otherwise, the air gap between the point of contact and the chip may lower the delivery efficiency and reduce the uniformity of distribution because the optimal use of the chip can only be achieved for flat surfaces. Optimization of intradermal electrode insertion position and depth is crucial for effective delivery.

• Apply an electric pulse through a reservoir containing the plasmid solution, using a needle array-based chip to create pores in the cell membrane for DNA entry.

• Use a square wave pulse with adjustable parameters via an electric pulse generator (CUY21EDIT II, Bex).

• Administer an initial poration pulse of 100 V to enhance cell membrane pore formation, followed by several driving pulses at the same voltage to drive plasmids into the tissue.

• Ensure voltages do not exceed 250 V to avoid cytotoxic effects.

Post-TNT characterization:

• Following TNT administration, confirm successful plasmid delivery by checking the Pd n list on the electroporator and expression by staining with appropriate antibodies and performing fluorescence microscopy imaging.

• Assess gene expression levels to ensure effective plasmid delivery into target cells using qPCR.

• Utilize super-resolution confocal microscopy (SRCM) to demonstrate the presence of exosomes with GFP reporter expression in both the epidermis and dermis.

This refined approach ensures efficient and effective plasmid delivery while minimizing potential cytotoxic effects, providing a robust method for gene delivery and expression studies in vivo.

Isolation of keratinocyte-derived exosomes (Exo_κ-GFP).

Because the GFP reporter protein was cloned in frame with CD9, CD63, and CD81, the pull-down of GFP using magnetic traps effectively isolates keratinocyte-derived exosomes (Exoκ-GFP) from the tissue homogenate, while non-keratinocyte derived exosomes (Exonon-GFP) were isolated from the flow-through using pan-CD supermagnetic beads. Keratinocyte-derived exosomes are then eluted from the GFP beads using an acidic elution buffer and neutralizing buffer. The resultant solution is ultracentrifuged for further purification of exosomes.

Proper pre-treatment of the reporter beads (GFP in this work), including washing and blocking, is critical to ensure the capture of specific exosome populations. The storage buffer of GFP beads contains 20% (v/v) ethanol that may interfere with the binding of exosomes and exosome functionality. Hence, washing with dilution buffer is a must. The beads must be blocked using 3% (w/v) bovine serum albumin (BSA) to avoid non-specific binding. This process takes 30–45 min. The separation method must be adapted to the tissue sample's complexity, focusing on the efficient release of exosomes without amphiosomic (intermediate organelles formed by the fusion of autophagosomes and endosomes) contamination. Once exosomes are suspended in PBS, adequate care should be taken not to vortex the exosome suspension to preserve membrane integrity and exosome functionality. Optimizing the above-mentioned steps is crucial for isolating exosomes without affecting their structure. The isolation procedure is most effective when performed on fresh tissue samples.

Characterization of exosomes:

• The size and concentration of Exoκ-GFP and Exonon-GFP are examined using NTA (Nanosight^™), as shown in Fig. 4a.

• Transmission/Scanning electron microscopy of Exoκ-GFP reveals the morphology of Exoκ-GFP, as shown in Fig. 2b.⁸⁵

• The presence of other exosomal markers can be tested using an exosome antibody array assay, as shown in Fig. 4b and Table 3.⁸⁴

• The presence of endosomal markers through flow cytometry analysis as shown in Fig. 4c.

Fig. 4 |. Characterization of keratinocyte-derived exosomes.

a, Representative particle size distribution of Exoκ-GFP by NTA from d5 WE murine tissue. (n=8). b, Exosome antibody array of d5 Exoκ-GFP from murine WE tissue. The labelled positive control is for HRP detection. c, Binding of RAB7AFITC and HSP90FITC with Exoκ-GFP. (n=6).

These findings demonstrate that the GFP-trap approach successfully isolates Exoκ-GFP from murine tissue homogenate. The data validates that the two-part isolation process effectively separates exosomes from the heterogeneous EV pool, providing a reliable method for isolating cell type-specific exosomes in vivo.

MATERIALS

Biological Materials

Primary cells

• C57BL/6J mouse primary epidermal keratinocytes (Cell biologics, cat. no. C57–6066k)

• C57BL/6J mouse primary dermal endothelial cells (Cell biologics, cat. no. C57–6064)

• C57BL/6J mouse primary dermal fibroblasts (Cell biologics, cat. no. C57–6067) CRITICAL All cells were maintained in a conventional culture incubator with 5% CO2 (v/v) humidified air. ! CAUTION All cells should be checked frequently to ensure they are authentic and free of mycoplasma contamination.

Animals

• Mice. For the results shown here, male C57BL/6J mice (The Jackson Laboratories; Strain no. 000664; RRID: IMSR_JAX:000664) aged 8–12 weeks were used. ! CAUTION Any animal experiments should be undertaken only after obtaining institutional regulatory board permission and must confirm to National and Institutional regulations. All experimental procedures in this protocol were approved by the IACUC of the University of Pittsburgh (Protocol No. 23083167). CRITICAL Ensure an adequate supply of food and water to the animals. The experiments must be performed in accordance with the circadian rhythm of the animal.

Reagents

Cell culture

• Complete epithelial cell medium with a kit (Cell biologics, cat. no. M6621)

• Complete endothelial cell medium with a kit (Cell biologics, cat. no. M1168)

• Complete fibroblast cell medium with a kit (Cell biologics, cat. no. M2267)

• 1X Dulbecco’s phosphate buffered saline; DPBS (Thermo Fisher Scientific-Gibco, cat. no. 14190–144)

• Fetal bovine serum, exosome depleted (FBS-Exo-D), one shot^™ format (Thermo Fisher Scientific, cat. no. A2720803)

Plasmid transformation

• Escherichia coli DH5α competent cells (Sigma-Aldrich, cat. no. CMC0007) CRITICAL The competent cells must be stored at −80 °C and thawed only before use. Unwanted thawing may result in reduced transformation efficiency.

• Luria-Bertani^{86, 87}, Low salt (Fisher Bioreagents, cat. no. BP1427–500) CRITICAL Luria-Bertani is hygroscopic; keep it at room temperature (RT).

• Recovery medium (or S.O.C. medium, super optimal broth with catabolite repression) (Sigma Aldrich, cat. no. CMR0002K).

• pLenti-Krt14-CD9-GFP Vector (ABM, cat. no. LV437001-Custom), pLenti-Krt14-CD63-GFP Vector (ABM, cat. no. LV485877-Custom) and pLenti-Krt14-CD81-GFP Vector (ABM, cat. no. LV457581-Custom)

• LB agar (Thermo Fisher Scientific-Invitrogen, cat. no. 22700–025)

• Kanamycin sulfate (Thermo Fisher Scientific-Gibco, cat. no. 11815024)

• Deionized double distilled water (In-house system-Milli-Q^® IQ-7000, Merck, Resistance: 18.2 Ω)

• UltraPure^™ distilled water DNAse, RNAse free (Thermo Fisher Scientific-Invitrogen, cat. no. 10977015)

• Ethyl alcohol (Fisher Scientific-Lab Alley Essential Chemicals, cat. no. EAS3C200– 5GAL)

• 50% (vol/vol) autoclaved Glycerol solution (Fisher Scientific, cat. no. BP229–1)

Plasmid amplification and isolation

• ZymoPURE^™ II plasmid midiprep kit (Zymo Research, cat. no. D4201)

• Pellet of the transformed E. coli DH5α cells containing desired plasmid DNA

• 25% (v/v) of glycerol stock of transformed DH5α for the desired plasmid

• Luria-Bertani, Low salt (Fisher Bioreagents, cat. no. BP1427–500)

• Molecular grade ethyl alcohol (Sigma Aldrich, cat. no. E7023–1L))

Plasmid validation

• 1X Dulbecco’s phosphate buffered saline; DPBS (Thermo Fisher Scientific-Gibco, cat. no. 14190–144)

• mirVana^™ miRNA isolation kit, with phenol (Thermo Fisher Scientific-Invitrogen, cat. no. AM1560)

• 5X VILO master mix (Thermo Fisher Scientific-Invitrogen, cat. no. 11754250)

• PowerUp SYBR^™ green PCR master mix (Thermo Fisher Scientific-Applied Biosystems, cat. no. A25742)

• mKrt14 forward primer 5’-GCTGGTGCAGAGCGGCAAGA-3’ (Integrated DNA Technologies, order no. 58940251)

• mKrt14 reverse primer 5’-AGACGGCGGTAGGTGGCGAT-3’ (Integrated DNA Technologies, order no. 58940252)

• mGapdh forward primer 5’-ATGACCACAGTCCATGCCATCACT-3’ (Integrated DNA Technologies, order no. 264242008)

• mGapdh reverse primer 5’-TGTTGAAGTCGCAGGAGACAACCT-3’ (Integrated DNA Technologies, order no. 264242009)

• Plasmids for labeling Exoκ-GFP. We successfully used Krt14 promoter-driven fluorescently GFP-labeled CD9, CD63, and CD81 encoding plasmids (backbone, pLenti, cat. no. LV437001-Custom, LV485877-Custom, and LV457581-Custom) constructed by Applied Biological Materials Inc., Richmond, BC, Canada.

• Lipofectamine^™ 3000 transfection reagent kit (Thermo Fisher Scientific-Invitrogen, cat. no. L3000015)

• Paraformaldehyde (PFA) 16% (w/v) aqueous solution (Fisher Scientific-Electron Microscopy Sciences, cat. no. 15710)

• Permeabilization buffer 10X (Thermo Fisher Scientific-Invitrogen, cat. no. 00–8333-56)

• Normal goat serum (NGS) blocking solution (Vector Laboratories, cat. no. S-1000)

• Tween^®20 (Sigma-Aldrich, cat. no. 8.17072.1000)

• Anti-GFP antibody (Abcam, cat. no. ab6556, RRID:AB_305564)

• Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor^™ 568 (Thermo Fisher Scientific, cat. no. A11011, RRID:AB_143157)

• 4',6-Diamidino-2-phenylindole, dihydrochloride (DAPI) (Thermo Fisher Scientific-Invitrogen, cat. no. D1306, RRID:AB_2869624)

Tissue nanotransfection (TNT) on cutaneous tissue

• Isoflurane (Patterson Veterinary, cat. no. 14043070405)

• 1X Dulbecco’s phosphate buffered saline; DPBS (Thermo Fisher Scientific-Gibco, cat. no. 14190–144)

• Plasmids of interest. We successfully used Krt14 promoter-driven fluorescent GFP-labeled CD9, CD63, and CD81 encoding plasmids (backbone, pLenti, cat. no. LV437001-Custom, LV485877-Custom and LV457581-Custom) constructed by Applied Biological Materials Inc., Richmond, BC, Canada.

• Deionized double distilled water (In-house system-Milli-Q^® IQ-7000, Merck, Resistance: 18.2 Ω)

• Ethanol wipes (Fischer Scientific, cat. no. 19–547-146)

• Betadine (Henry Schein, cat. no. 67618–151-17)

• Nairing cream (Karewell Brands Inc, cat. no. 22339)

• Exfoliating cream (Amazon, cat. no. B002HS42UC)

Wounding procedure on mice

• Isoflurane (Patterson Veterinary, cat. no. 14043070405)

• Betadine (Henry Schein, cat. no. 67618–151-17)

Euthanasia and tissue harvesting

• 10% Neutral buffered formalin (v/v) (Azer Scientific, cat. no. NBF-4-G)

• Optimal cutting temperature compound (Fischer Scientific, cat. no. 23–730-571)

• Dry ice

• Liquid nitrogen (LN2, institutional)

Quantifying gene expression

• 5X VILO master mix (Thermo Fisher Scientific-Invitrogen, cat. no. 11754250)

• PowerUp SYBR^™ green PCR master mix (Thermo Fisher Scientific-Applied Biosystems, cat. no. A25742)

• mirVana^™ miRNA isolation kit, with phenol (Thermo Fisher Scientific-Invitrogen, cat. no. AM1560)

• Liquid nitrogen (LN2, institutional)

• RNaseZap^™ RNase decontamination solution (Thermo Fischer Scientific-Invitrogen, cat. no. AM9780)

• Molecular grade ethyl alcohol (Sigma Aldrich, cat. no. E7023–1L))

Immunocytochemistry and immunohistochemistry

• 10X Phosphate buffered saline, PBS (Fisher Scientific, cat. no. BP39920)

• Acetone (Sigma-Aldrich, cat. no. 534064) ! CAUTION Acetone is highly volatile, and flammable. It must be handled in a fume hood.

• Hard set mounting media (Vector Laboratories, cat. no. H-1400)

• Normal goat serum (NGS) blocking solution (Vector Laboratories, cat. no. S-1000)

• 4',6-Diamidino-2-phenylindole, dihydrochloride (DAPI) (Thermo Fisher Scientific-Invitrogen, cat. no. D1306)

• Anti-GFP antibody (Abcam, cat. no. ab6556, RRID:AB_305564)

• Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor^™ 568 (Thermo Fisher Scientific, cat. no. A11011, RRID:AB_143157)

• Goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor^™ Plus 405 (Thermo Fisher Scientific, cat. no. A48255, RRID:AB_2890536)

• M.O.M^® (Mouse on Mouse) blocking reagent (Vector Laboratories, cat. no. MKB-2213– 1)

Isolation of exosomes

• 1X Dulbecco’s phosphate-buffered saline; DPBS (Thermo Fisher Scientific-Gibco, cat. no. 14190–144)

• ChromoTek GFP-Trap^® magnetic agarose (Proteintech, cat. no. gtma-20)

• Glycine, 0.2 M buffer solution, pH 3.0 (Thermo Fisher Scientific, cat. no. J62567.AP)

• Tris (1 M), pH 8.0, RNase-free (Thermo Fisher Scientific-Invitrogen, cat. no. AM9856)

• UltraPure^™ 0.5 M EDTA, pH 8.0 (Fisher Scientific-Invitrogen, cat. no. 15–575-020)

• Sodium Chloride (Fisher Scientific, cat. no. S271–500)

• UltraPure^™ distilled water DNAse, RNAse free (Thermo Fisher Scientific-Invitrogen, cat. no. 10977015)

• Bovine serum albumin (BSA) (Sigma-Aldrich, cat. no. A4612)

• Ammonium hydroxide solution (Millipore Sigma, cat. no. 221228)

Nanoparticle tracking analysis (NTA)

• 100 nm, and 200 nm polystyrene latex beads (Malvern Panalytical, cat. no. NTA4091)

• UltraPure^™ distilled water DNAse, RNAse free (Thermo Fisher Scientific-Invitrogen, cat. no. 10977015)

Transmission electron microscopy (TEM)

• Sodium cacodylate buffer pH 6.5; 50 mM – Isopropanol 15% (v/v) solution (Sigma-Aldrich, cat. no. 97068) ! CAUTION Sodium cacodylate is toxic if ingested, inhaled or absorbed in skin, so wear personal protective equipment and handle it with care.

• Uranyl acetate solution 1% (w/v) and 2% (w/v) (Electron Microscopy Sciences, cat. no. 22400) ! CAUTION Uranyl acetate is carcinogenic, so wear personal protective equipment and handle it with care.

• Paraformaldehyde (PFA), 4% (w/v) in PBS (Biotium, cat. no. 22023) ! CAUTION Paraformaldehyde is flammable, carcinogenic, and toxic. It is ONLY meant to be used under the fume hood.

• Ammonium chloride (Millipore Sigma, cat. no. 193621)

• 10X Phosphate-buffered saline, PBS (Fisher Scientific, cat. no. BP39920)

Exosome antibody array assay

• 1X Dulbecco’s phosphate-buffered saline; DPBS (Thermo Fisher Scientific-Gibco, cat. no. 14190–144)

• Protease inhibitor cocktail (PIC) (Sigma-Aldrich, cat. no. P8340)

• Phenylmethylsulfonyl fluoride (PMSF) (Thermo Fisher Scientific, cat. no. 36978)

• 10X Cell lysis buffer (Cell Signaling Technology, cat. no. 9803)

• Pierce^™ BCA protein assay kit (Thermo Fisher Scientific, cat. no. 23225)

• Exosome antibody array kit (System Biosciences, cat. no. EXORAY400A-8)

• UltraPure^™ distilled water DNAse, RNAse free (Thermo Fisher Scientific-Invitrogen, cat. no. 10977015)

• Developer (Azure Biosystems, cat. no. AC2103)

• Femtogram HRP substrate (Radiance Plus) enhancer (Azure Biosystems, cat. no. AC2103)

• Tween^® 20 (polysorbate) (Millipore Sigma, cat. no. 817072)

• Electrochemiluminescence (ECL) (Advansta, cat. no. K-12047-D50)

Flow cytometry

• Sheath additive (BD biosciences, cat. no. 660584)

• UltraPure^™ distilled water DNAse, RNAse free (Thermo Fisher Scientific-Invitrogen, cat. no. 10977015)

• Detergent solution concentrate (BD biosciences, cat. no. 660585)

• BD FACSClean (BD Biosciences, cat. no. 340345)

• CF Dyes using Mix-n-Stain Antibody Labeling Kits (Biotium, cat. no. 92273)

• Anti-HSP90 (Abcam, cat. no. ab59459, RRID:AB_942030)

• Anti-RAB7A (Abcam, cat. no. ab137029, RRID:AB_2629474)

Sucrose density gradient ultracentrifugation of exosomes

• 1X Dulbecco’s phosphate-buffered saline; DPBS (Thermo Fisher Scientific-Gibco, cat. no. 14190–144)

• Sucrose (Fisher Scientific, cat. no. S5–500)

• UltraPure^™ distilled water DNAse, RNAse free (Thermo Fisher Scientific-Invitrogen, cat. no. 10977015)

Equipment

Plasmid transformation

• Aervent^®-50 filter unit 0.22 μm disposable filter (Millipore Sigma, cat. no. MTGR75010)

• Fisherbrand^™ reusable glass wide-mouth Erlenmeyer flasks, capacity: 1000 ml (Fisher Scientific, cat. no. FB5011000).

• Polymethylpentene graduated cylinders, capacity: 100 ml (Fisher Scientific, cat. no. 03– 007-33); 250 ml (Fisher Scientific, cat. no. 03–007-34) and 1000 ml (Fisher Scientific, cat. no. 03–007-36)

• FALCON^®14 ml polypropylene round-bottom tube with cap, for bacterial culture, non-pyrogenic, 17 × 100 mm style (Corning, cat. no. 352059)

• Sterile 2.0 ml cryogenic vials (Corning, cat. no. 430661)

• Aseptic petri dishes with lid (Bacteriological, cat. no. 08–757-100D)

• Sterile polystyrene disposable serological pipet with magnifier stripe, 5 ml, 10 ml, 25 ml (Fisher Scientific, cat. no. 13–678-11D, 13–678-11E, 13–678-11)

• Fisherbrand^™ Elite Pipette Kit Pipettes^® (Fisher Scientific, cat. no. 14–388-100, Set of 4 pipettes 0.2–2 μl, 2–20 μl, 20–200 μl, 100–1000 μl)

• Low binding barrier pipet tips, sterile, polypropylene, graduated 0.1–10 μl (Corning, cat. no. 4119)

• ART^® 200 barrier tip 200 μl pipette tips, (Thermo Fischer Scientific, cat. no. 2069–05-HR)

• Pipet tips, sterile, polypropylene, graduated 100–1000 μl (Corning, cat. no. 4119)

• Disposable cuvettes (Fisher Scientific, cat. no. 14–955-127)

• L-shaped cell spreaders (Fisher Scientific, cat. no. 14–665-230)

• Nitrile examination, powder-free gloves (HandPRO, cat. no. 1102)

• Weighing balance (Adventurer^™ OHAUS, model no. AR5120). CRITICAL Calibration and taring of the weighing balance is necessary before use.

• Laminar air hood cabinet (LABCONCO A2, model no. 302411101)

• Bacterial incubator at 37 ºC (Thermoscientific, model no. RF88532)

• Water bath (Thermoscientific, model no. TSGP05)

• Freezer, −80 °C (Thermoscientific, model no. 817CD)

• Freezer, −20 °C (LabRepCo, model no. 085221)

• Refrigerator, 4 °C (General Electric Company, model no. GMR04AAMBWW)

• UV-Visible (UV-Vis) spectrophotometer (Thermo Fisher Scientific, model no. NANODROP One^c)

• Ice maker (Scotsman, model no. UF424A-1A)

Plasmid amplification and isolation

• High-density polyethylene (HDPE) coated wire racks (Fisher Scientific, cat. no. FB147916A)

• 50 ml polypropylene falcon^® conical tube, (Corning, cat. no. 14–959-49A)

• 1.5 ml microcentrifuge tube with locking lid, sterile (Fisher Scientific, cat. no. 14–666-319)

• Autoclave LV 250 laboratory steam sterilizer (STERIS^®, Amsco^®, Lab 250)

• Microcentrifuge (Thermo Fisher Scientific, model-Fisher AccuSpin Micro 17R)

• UV-Visible (UV-Vis) spectrophotometer (Thermoscientific, NANODROP One^c)

Plasmid validation

• Nunc^™ cell-culture treated multidishes (Thermo Fisher Scientific, cat. no. 150628)

• Cell lifter (Corning, cat. no. 3008)

• MicroAmp^™ optical 8-cap strip (Thermo Fisher Scientific-Applied Biosystems, cat. no. 4323032)

• MicroAmp^™ fast optical 96-well reaction plate with barcode, 0.1 ml (Thermo Fisher Scientific-Applied Biosystems, cat. no. 4346907)

• MicroAmp^™ optical adhesive film (Thermo Fisher Scientific-Applied Biosystems, cat. no. 4311971)

• 35 mm collagen-coated dishes no. 1.5 coverslip, 14 mm glass diameter (Mattek, cat. no. P35gcol-1.5–14-c)

• Accuspin micro 17R (Thermo Fisher Scientific, cat. no. 2023–03-21)

• Simpliamp thermal cycler (Applied Biosystems, cat. no. 24812)

• QuantStudio 3 (Applied Biosystems, cat. no. A28131)

• Super-resolution confocal microscope (Zeiss, model no. LSM 880)

Tissue nanotransfection (TNT)

• Euthanasia chamber-100% CO2 at a fill rate of 30–70% chamber volume per min

• BD precision glide hypodermic needles 26 G needle (Fisher Scientific, cat. no. BD30511)

• 3M Scotch tape (Vavantor by VWR, cat. no. 76247–202)

• Hair trimmer (Kent Scientific Corporation, cat. no. CL7300-Kit)

• SomnoSuite low-flow anesthesia system (Kent Scientific, model no. SS-01)

• Pulse generator (Bex, model no. CUY21EDIT II)

• 2 hook clip lead cables (Amazon, cat. no. B07TF8K9NC)

• Gold-plated electrode (100 μl Neon^® Tip, part of kit MPK10025)

• TNT chip (in house)

• Ear tags (Fisher Scientific, cat. no. 50–486-427)

• Braintree Scientific Small Animal Ear Tag Applicator Only (Fisher Scientific, cat. no. NC9014296)

Wounding procedure on mice

• SomnoSuite low-flow anesthesia system (Kent Scientific, model no. SS-01)

• Acuderm Inc Acuderm Acu-Punch Biopsy Punch, Sterile, 10 mm, (Fisher Scientific, cat no. NC9236770)

• Acuderm Inc Acu-Punch; 12mm; Acuderm (Fisher Scientific, cat no. NC9253254)

• Acuderm Inc 8mm Biopsy punches PK/25 (Fisher Scientific, cat no. NC9324386)

• World Precision Instrument Cutting Mats for Biopsy Punches-504621 (Fisher Scientific, cat no.50–253-9324)

• Microsurgery kit for mice and rats (Animalab)

• Fine forceps (Fine Science Tools, cat. no. 11150–10)

Euthanasia and tissue harvesting

• Sterile disposable towel drapes (Kent Scientific Corporation, cat. no. SURGI-5023)

• Surgical design disposable scalpels (Fischer Scientific, cat. no. 22–079-707)

• Biopsy punches (Fischer Scientific, cat. no. NC9324386)

• 2 ml external threaded polypropylene cryogenic vial with round bottom (Corning, cat. no. 430661)

• Eisco^™ clear safety glasses, vented (Fischer Scientific, cat. no. S41140)

• Tempshield^™ cryo-gloves^™ blue mid-arm length cryogenic gloves (Fischer Scientific, cat. no. S47320)

• Epredia^™ cassette ii slotted tissue cassettes in tube packs (Fischer Scientific, cat. no. B851729GN)

• Biopsy sponge pads for cassettes (Fischer Scientific, cat. no. NC0259333)Tissue-Tek^® cryo-mold (Electron Microscopy Sciences, cat. no. 62534)

• Scissors (Fine Science Tools, cat. no. 14501–14)

• Fine scissors (Fine Science Tools, cat. no. 14558–11)

• Fine forceps (Fine Science Tools, cat. no. 11150–10)

• Benchtop liquid nitrogen containers (Thermo Fischer Scientific, cat. no. 2123)

• World Precision Instrument Cutting Mats for Biopsy Punches-504621 (Fisher Scientific, cat no.50–253-9324)

Quantifying gene expression

• MicroAmp^™ fast optical 96-well reaction plate with barcode, 0.1 ml (Thermo Fisher Scientific, cat. no. 4346906)

• Optical adhesive covers (Thermo Fisher Scientific, cat. no. 4360954)

• 0.2 ml flat cap individual tubes (Thermo Fisher Scientific, cat. no. AB0620)

• Eisco^™ thick-walled mortar and pestle set (Fisher Scientific, cat. no. S39833)

• Scienceware^® polyethylene dipper (Sigma-Aldrich, cat. no. Z206725–1EA)

• 2.0 ml safe-lock tubes, natural, PCR clean (Eppendorf, cat. no. 022363344)

• 1.5 ml microcentrifuge tube with locking lid, sterile (Fisher Scientific, cat. no. 14–666-319)

• Liquid nitrogen (LN2, institutional)

• Thermo-Flask^™ benchtop liquid nitrogen containers (Thermo Fisher Scientific, cat. no. 2123)

• Fisherbrand^™ polyurethane ice buckets (Fisher Scientific, cat. no. 02–591-45)

• Lysis homogenizer bead (Benchmark Scientific, cat. no. D1033–28)

• Pipettes^® (Fisher Scientific, cat. no. 14–388-100)

• TissueLyser III (QIAGEN, model no. 9003240)

• Weighing balance (Denver Instrument, model no. M-120)

• Microcentrifuge accuSpin micro17 (Thermo Fisher Scientific, cat. no. 13100675)

• Heat block (Thermo Fisher Scientific, cat. no. 88870001)

• Vortex mixer (Thermo Fisher Scientific, model no. 88882011)

• Bench-top spinner, 0.2 ml tubes, (Benchmark Scientific Inc., model no. StripSpin^™ 12 C1012)

• Microplate centrifuge (Fisherbrand, model no. 14955300).

• UV-Visible (UV-Vis) spectrophotometer (Thermoscientific, NANODROP One^c)

• Simpliamp thermal cycler (Applied Biosystems, cat. no. 24812)

• QuantStudio 3 (Applied Biosystems, cat. no. A28131)

Immunocytochemistry and Immunohistochemistry

• Slide staining jar (Electron Microscopy Sciences, model no. 71385)

• Slide staining rack (Electron Microscopy Sciences, model no. 71386-DR)

• ImmEdge hydrophobic barrier PAP pen (Vector Labs, cat. no. H-4000)

• Cryomolds disposable base (Fisher Scientific, cat. no. 22–363-556)

• Cover glasses superfrost (Fisher Scientific, cat. no. 12–542B)

• Superfrost plus microscope slides (Fisher Scientific, cat. no. 1255015)

• Extremus microtome blades (Fisher Scientific, C.L. Sturkey, cat. no. 22210033)

• Cryostat with a razor (Thermoscientific, model no. CryoStar NX50)

• Super-resolution confocal microscope (Zeiss, model no. LSM 880)

Isolation of exosomes

• 1.5 ml microcentrifuge tube with locking lid, sterile (Fisher Scientific, cat. no. 14–666-319).

• Eisco^™ clear safety glasses, vented (Fischer Scientific, cat. no. S41140)

• Tempshield^™ cryo-gloves^™ blue mid-arm length cryogenic gloves (Fischer Scientific, cat. no. S47320)

• Eisco^™ thick-walled mortar and pestle set (Fisher Scientific, cat. no. S39833)

• Scienceware^® polyethylene dipper (Sigma-Aldrich, cat. no. Z206725–1EA)

• Ultracentrifuge tube, 1 ml, open-top thickwall polycarbonate tube, 11 × 34 mm − 100Pk (Beckman Coulter, cat. no. 343778)

• Weighing balance (Adventurer^™ OHAUS, model no. AR5120)

• Microcentrifuge accuSpin micro17 (Thermo Fisher Scientific, cat. no. 13100675)

• Vortex mixer (Thermo Fisher Scientific, model no. 88882011)

• Rotator (Benchmark, model no. Rotator-MiNi, cat. no. R2020)

• Ultracentrifuge (Beckman Coulter^®, model no. Optima^™ MAX-XP ultracentrifuge)

• Rotor-TLA 120.2 120K RPM (Beckman Coulter, serial no. 18U2067)

• MiniMACS^™ separator (Miltenyi Biotec, cat. no. 130–042-102)

• Fine forceps (Fine Science Tools, cat. no. 11150–10)

Nanoparticle tracking analysis (NTA)

• BD disposable syringes with luer-lok^™ tips (Fisher Scientific, cat. no. 14–823-30)

• NanoSight Pro (Malvern Panalytical, model no. HBG5000)

• NanoSight Pro laser module (Malvern Panalytical)

• Syringe pump Malvern OEM module (Malvern Panalytical, model no. 986778)

Zeta potential (ζ)

• BD disposable syringes with luer-lok^™ tips (Fisher Scientific, cat. no. 14–823-30)

• Folded Capillary Zeta Cell (Malvern Panalytical, part no. DTS1070 cell)

• Zetasizer nano (Malvern Panalytical Limited, model no. ZEN2600)

Transmission electron microscopy (TEM)

• 300 μm carbon-coated copper grid (Electron Microscopy Sciences, cat. no. FCF300-Cu-50)

• Carbon-formvar coated 200 mesh nickel grids (Electron Microscopy Sciences, cat. no. FCF200-Ni-50)

• TEM grid holder block (Ted Pella, cat. no. 16820–25)

• TEM single tilt holder (Thermo Fisher Scientific, cat. no. MOTILTROTSAMPLHOLD)

• PELCO easiGlow glow discharge cleaning system (Ted Pella, cat. no. 91000)

• Transmission electron microscope (JEOL, model no. JEM-1400plus)

Exosome antibody array assay

• Nunc^™ microwell^™ 96-well microplates (Thermo Fisher Scientific, cat. no. 269787)

• 1.5 ml microcentrifuge tube with locking lid, sterile (Fisher Scientific, cat. no. 14–666-319)

• Incubation boxes (Azure systems, cat. no. AC2120)

• Synergy HTX multi-mode reader (BioTek, model no. Synergy HTX)

• Incubator at 37 ºC (Thermo Fisher Scientific, model no. RF88532)

• Microcentrifuge accuSpin micro17 (Thermo Fisher Scientific, cat. no. 13100675)

• Membrane imager (Azure Biosystems, model no. Azure 600, cat. no. AZI600–01)

• Vortex mixer (Thermo Fisher Scientific, model no. 88882011)

• Analog rocking platform shaker single-tier (VWR, cat. no. 10127–872)

• UV-vis spectrophotometer (Nanodrop)

Flow cytometry

• 1.5 ml microcentrifuge tube with locking lid, sterile (Fisher Scientific, cat. no. 14–666-319)

• BD Accuri (BD Biosciences, model no. BD Accuri C6 Plus)

Sucrose density gradient ultracentrifugation of exosomes

• 1.5 ml microcentrifuge tube with locking lid, sterile (Fisher Scientific, cat. no. 14–666-319)

• Ultracentrifuge tube, 1 ml, open-top thick wall polycarbonate tube, 11 × 34 mm − 100Pk (Beckman Coulter, cat. no. 343778)

• Ultracentrifuge (Beckman Coulter^®, model no. Optima^™ MAX-XP ultracentrifuge)

• Rotor-TLA 120.2 120K RPM (Beckman Coulter, serial no. 18U2067)

Softwares

• FlowJo (V_10.10); https://www.flowjo.com/flowjo/download

• Zen Blue (V_3.8); https://www.zeiss.com/microscopy/us/products/software/zeiss-zen-lite.html

• NS Xplorer software (V_1.1.0.6); https://www.malvernpanalytical.com/en/support/product-support/software/nanosight-pro-software-update-v1-0

• Zetasizer software (V_8.02); https://www.malvernpanalytical.com/en/support/product-support/software/zetasizer-family-software-update-v8-02

• Graph Pad (V_10.2.2); https://www.graphpad.com/search/?searchquery=download

• Image J; https://imagej.en.softonic.com/download

• Randomization; https://www.random.org/

Reagent Set up

CRITICAL All reagents must be prepared fresh unless mentioned otherwise.

Plasmid transformation

• Preparation of kanamycin sulfate

  1. Prepare 50 mg/ml stock solution of kanamycin sulfate using ultrapure water. Filter the dissolved kanamycin sulfate solution using a sterile membrane filter (0.22 μm filter) in a biosafety cabinet and store aliquots at −20 °C until further use.
• Preparation of LB agar plates

  1. Weigh 8 g LB Agar (LBA) and dissolve in 250 ml of double distilled water.
  2. Autoclave the prepared LBA solution at 121°C and 15 psi for 15 min.
  3. Following autoclave, wait until the temperature of the agar media drops down to 50–55 °C.
  4. Take the LB agar to the biosafety cabinet and add 250 μl of kanamycin sulfate (50 μg/ml working concentration). ▲CRITICAL STEP The LB agar temperature must cool down to around 50 °C before adding the antibiotics. Excessive heat will degrade the antibiotic.
  5. Gently swirl the medium containing the antibiotic and then pour 20 ml into each sterile petri plate. ! CAUTION To avoid bubble formation, the residual drops of LBA on the edges of the petri plate should be dispersed with a sterile pipet tip.
  6. Allow plates to solidify for 15–20 min in the biosafety cabinet. These solidified petri plates containing LBA medium will be used for bacterial transformation.

■PAUSE POINT The plates can be stored at 4 °C in a sealed plastic bag until further use.

• Preparation of LB broth media

  1. Dissolve 5 g of LB in 250 ml of double distilled water in 500 ml capacity conical flasks for growing the transformed E. coli DH5α cells. CRITICAL This is for one plasmid only. For the cocktail of three plasmids, scale up accordingly.
  2. Autoclave it at 121 °C and 15 psi for 15 min. ■PAUSE POINT The LB broth can be kept at 4 °C/RT until further use for a few days.
  3. Add 250 μl of kanamycin sulfate (50 μg/ml working concentration) and just before use, add antibiotics to the LB media at RT.

Isolation of exosomes

• Wash buffer/dilution buffer: 10 mM Tris/Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA

  1. In a 50 ml centrifuge tube, combine 49.5 ml UPW and 500 μl of 1M Tris/Cl to make a final solution of 10 mM Tris/Cl.
  2. Weigh 438 mg of NaCl (F.W. 58.44 g/mol) and add to tube, cap, and shake to dissolve to make a final solution of 150 mM NaCl.
  3. Weigh 7.3 mg EDTA (F.W. 292.25 g/mol) and add to tube, cap, and shake to dissolve to make a final solution of 0.5 mM EDTA.
  4. Cool at 4 °C or on ice for 1 h.
  5. Calibrate the pH meter with basic, neutral, and acidic standards.
  6. Adjust pH of the solution at 4 °C. Using a calibrated pH meter in solution and fume hood, adjust to pH 7.5 by adding 1 μl at a time of 30% ammonium hydroxide to the 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA in UPW solution (you can probably use NaOH but note this will slightly alter the [Na⁺].
  7. If you increase the pH beyond 7.5, use glacial acetic acid in a similar manner mentioned above to correct the pH.

• Blocking buffer: 3% (w/v) bovine serum albumin (BSA). In a 50 ml centrifuge tube, combine 50 ml UPW and 1.5 g of BSA. Swirl gently to make a homogeneous solution. ! CAUTION Excessive stirring will make the solution frothy. To avoid froth, the solution can be prepared the previous day and stored at 4 °C.

• Acidic elution buffer: 200 mM glycine pH 3.0

• Neutralization buffer: 1 M Tris pH 8.0.

Nanoparticle tracking analysis

• Dilute the exosome samples with UPW (1:100–1:1000) before running NTA, depending on the concentration of the exosomes⁵³.

Zeta Potential

• Dilute the exosome samples with UPW (1:50–1:100) depending on the concentration of the exosomes.

Transmission electron microscopy

• Use the 0.2 M sodium cacodylate buffer as per the manufacturer’s directions.

• Prepare 16% (w/v) of PFA in UPW.

• Prepare the TEM reagents with caution in the sequence as described in the procedure steps later. ! CAUTION Use suitable protections due to the inherent carcinogenicity and radioactivity of these compounds.

Exosome antibody array assay

• Prepare all reagents following the manufacturer’s instructions provided with the kit.

• For protein estimation, the BCA reagent A ((x * 200) + 500 μl; × is the no. of wells) and BCA Reagent B ((Amount of reagent A)/50 μl) should be combined to make the working reagent. CRITICAL The 50:1 ratio of reagent A and reagent B is important. This ratio ensures the optimal formation of the purple-colored complex that forms when the protein reacts with the working reagent so the complex can be detected at 562 nm.

Flow cytometry

• To prepare the sheath solution, combine 10 ml of sheath additives with 1 L of UPW.

• Prepare a detergent solution by adding 3 ml of detergent solution concentrate in 197 ml UPW.

• FACS clean solution is provided by the manufacturer.

Sucrose density gradient ultracentrifugation of exosomes

• Prepare sucrose solutions by dissolving sucrose in 1 mM PBS to make 0.5 M, 1 M, 1.5 M, and 2.5 M solutions. The final volume of each sucrose solution should be 250 μl. CRITICAL It is imperative to make fresh sucrose solutions each time.

Equipment Set up

Ultracentrifugation

• Set the rotor (Beckman Coulter) with the ultracentrifuge tubes containing samples into the centrifuge and rotate it clockwise until you hear a click. ! CAUTION Using the wrong rotor or setting it incorrectly in the ultracentrifuge can damage the ultracentrifuge.

• Press the lock of the rotor present at the centre from the top.

Cryostar NX50

• Before sectioning, ensure that the blade holder and cryo chamber are steady and near the specified cutting temperature.

• All equipment that comes into contact with the sections or is used to treat the specimen must likewise be chilled to prevent the tissue from attaching to it.

• Keep all the accessories on shelves or in the brush tray in the cryo chamber.

• To prevent warm air from entering the cryo chamber, keep the window closed during inactivity and specimen preparation. ! CAUTION Only trained personnel should perform cryosection using the cryostat to avoid injuries and accidents.

Nanoparticle tracking analysis

• Calibrate the NanoSight Pro machine with 100 nm and 200 nm polystyrene latex beads to optimize detection settings recorded by a highly sensitive camera.

• To configure the parameters for exosome samples, use the following settings: The temperature is set to 24.2 °C.

  The single capture duration is 21.9 s. The filter used is a light scatter.

  The flow rate is 4 μl/min. The number of captures is 10. Viscosity is 0.9053 cP.

  The frames captured is 750.

Zeta potential

• After turning the machine on, make sure the temperature is at 25 °C. CRITICAL Keep the Zetasizer lid closed and wait up to 30 min for the laser to stabilize. This is useful in avoiding thermal equilibration problems that may affect the results.

Exosome antibody array assay

• 37 °C incubator: Turn on the incubator and set the temperature to 37 °C. CRITICAL This must be done ahead of time (at least 15–20 min before) as the incubator needs time to rise to 37 °C.

• Synergy HTX multi-mode reader: Turn on the plate reader and make sure the protein estimation protocol is selected. ! CAUTION You will not get accurate results if the protocol is set to anything other than 562 nm wavelength of light. It is imperative to ensure the correct protocol has been selected.

Azure biosystems C600 imager

• Turn the system on and select chemiluminescence and color marker. After this, select auto image, set exposure time (optimized by the user), and image. ▲CRITICAL Manual imaging is recommended, as you can set different exposure times.

Flow cytometry

• Ensure that the sheath solution, detergent solution, and FACSclean bottles are full before turning the system on. There should be 2 L of sheath solution, 120 ml of detergent solution, and 250 ml of BD FACSClean solution in the working bottle of the instrument. The waste bottle should have 200 ml of undiluted bleach. ! CAUTION If the reagent amounts are less than the specified volumes, then the system will suck up air which can interfere with the detection of events.

• Make sure the injection probe is dipped in at least 2 ml of UPW before and after the completion of the experiment.

• After turning the instrument on, the cytometer fills the fluid lines with sheath fluid for about 15 min. As the laser warms up the indicator flashes yellow. CRITICAL The lid of the cytometer must remain closed while the laser is heating up. This is imperative as opening the lid will interfere with the heating process and cause delays in running samples.

• Before shutting down the system, dip the injection probe into BD FACSClean solution for Sample Injection Port (SIP) clean. The fluidics clean cycle lasts approximately 15 min. ! CAUTION Shutting down the program incorrectly results in delayed start-up time.

Procedure

Labeling and isolation of exosomes of keratinocyte origin

Transformation of plasmid DNA in E. coli DH5α competent cells ● Timing 2 d

• 1Thaw the competent cells stored at −80 °C by placing the vials on ice for 5–10 min.
• 2Vortex and spin down the plasmids at 2000 × g for 10 s at RT.
• 3Add 4 μl of desired plasmid (400 ng) in a round-bottom polypropylene tube.
• 4Add 40 μl of E. coli DH5α competent cells on top of the plasmids and incubate the tubes for 30 min on ice. ! CAUTION Do not mix the plasmid and competent cells by pipetting.

  ▲CRITICAL STEP In one empty round-bottom polypropylene tube, just add 40 μl of E. coli DH5α competent cells as a negative control.

• 5To heat shock the cells, place them in a 42 °C water bath and gently shake for 30 s. !

  CAUTION The cells must not be shaken vigorously during heat shock, as heat shock itself is a harsh treatment for bacterial cells. ▲CRITICAL STEP The heat shock must be given at 42 °C for a minimum time of 30 s and a maximum time of 45 s. Longer exposure of competent cells to the heat shock (>60–90s), may lead to loss of competence by the bacterial cells and, hence, colonies will not be observed due to failed transformation. Always use a fresh stock of competent cells stored at −80 °C.

• 6Place the cells instantly on ice for 2 min.
• 7Gently add 360 μl of recovery media (SOC) to the tubes and place them in a shaker incubator at 225 rpm for 1 h at 37 °C.
• 8Label the LB agar plates with respective sample names or negative control.
• 9Following the 1 h incubation, add 10 μl of the transformed cells on the LB agar plates (prepared with kanamycin sulfate) and spread uniformly using a spreader. ! CAUTION Do not discard the remaining transformed cells; they can be stored at 4 °C and used on the next day if desired. ▲CRITICAL STEP Always prepare negative controls. For rigor, it is prudent to include one plate without any cells (to check for contamination of LB agar plates) and another with the control untransformed competent cells only (to check for kanamycin selection).
• 10Incubate the plates at 37 °C in a bacterial incubator overnight (~12–16 h) in an inverted position. ! CAUTION Let the inoculum completely absorb the agar on the LBA plate before inverting the plates to avoid the smeary growth of bacteria.
• 11Observe the LB agar plates the next day for the growth of plasmid-transformed bacteria (Fig. 5a).
• 12From each plate with plasmid-transformed bacteria, pick a single colony of E. coli DH5α (Fig. 5a) and inoculate it in a round-bottom culture tube containing 5 ml LB broth and a final concentration of desired antibiotics (50 μg/ml working concentration of kanamycin sulfate).

  ?TROUBLESHOOTING

• 13Incubate the colony picked from step 12 in a 5 ml as a day culture in a shaker incubator at 180–220 rpm for 4–6 h at 37 °C (Fig. 5b).
• 14Transfer 1 ml of the culture to a 1 ml disposable cuvette.
• 15Measure the culture's optical density (OD) at 600 nm (OD600) against LB broth using a UV-Vis spectrophotometer (NanoDrop One). If the OD600 is ~0.2–0.3, then inoculate the transformed cells for plasmid amplification as described in Step 16. You can also prepare glycerol stocks of the transformed cells by mixing 500 μl of culture and 500 μl of autoclaved 50 % (v/v) glycerol in a 2.0 ml cryogenic vial and store at −80 °C until further use. ! CAUTION Keep on checking the OD600 of the day culture so as not to exceed 0.2– 0.3.

Fig. 5 |. Transformation of plasmid construct for labeling and isolation of keratinocyte-derived exosomes in competent cells.

a, Growth of kanamycin-resistant bacterial colonies on agar plates post-transformation. No bacteria were plated on the negative control agar plate, serving as a control for contamination, and the ‘DH5α control’ plate was inoculated with untransformed E. coli DH5α cells to control for kanamycin selection. Shown on the right is a plate inoculated with plasmid transformed DH5α cells. The inset image shows a zoomed image of a single colony (black arrowhead) of transformed DH5α cells using a Krt14-CD-GFP plasmid cocktail. Scale, 50 mm b, Growth of transformed DH5α from a single colony in LB media after day culture at 37 °C. Left: negative control (LB media); right: transformed DH5α with Krt14-CD-GFP plasmids cocktail.

Plasmid amplification and isolation ● Timing 20 h (including 16 h overnight culture)

• 16Inoculate 250 μl of transformed E. coli DH5α cells from Step 13 in 250 ml LB broth with 250 μl of kanamycin sulfate (50 μg/ml working concentration).
• 17Incubate the culture overnight (~16 h) in a shaker incubator at 37 °C at 180–220 rpm.
• 18After overnight incubation, transfer 1 ml of the culture to a 1 ml disposable cuvette.
• 19Measure the culture's OD600 (value must be ~0.8–1.0) against LB broth using a UV-Vis spectrophotometer (NanoDrop One).
• 20Distribute 250 ml bacterial culture into five 50 ml Falcon (conical) tubes and centrifuge at 3400 × g for 10 min at 37 °C to pellet the cells. ■PAUSE POINT The cell pellet can be stored at −80 °C until further use.
• 21Isolate the plasmids as specified in the manufacturer’s protocol given with the ZymoPURE^™ II plasmid midiprep kit.

Promoter validation ● Timing 30 h

• 22Seed 1x10⁶ primary mouse keratinocytes, fibroblast, and endothelial cells per well in three different 12-well plates using medium of cells as mentioned in the reagents section.
• 23After 24 h, aspirate the medium and wash the cells thrice with 500 μl to 1 ml sterile cell culture PBS per well.
• 24Add 250 μl of PBS per well and scrape the washed cells using cell scrapers. Pool the cells from each plate into a 15 ml conical tube.
• 25Centrifuge the scraped cells at 500 × g for 5 min at 4 °C.
• 26Aspirate the PBS, collect and store the cell pellets or proceed immediately to cell lysis.

  ■PAUSE POINT Samples can be stored at −80 °C indefinitely and further processed later.

• 27Add 300 μl of lysis buffer from the mirVana Total RNA Isolation Kit.
• 28Extract the RNA using the mirVana Total RNA Isolation Kit following the manufacturer's protocol. ! CAUTION The mirVana Total RNA Isolation Kit is often preferred over Trizol reagent for RNA isolation, due to its higher RNA recovery and less impact on RNA integrity. While Trizol is effective for total RNA isolation, it can sometimes lead to lower purity and potential degradation.
• 29Measure the concentration of RNA using nanodrop. ▲CRITICAL STEP The ratio of 260/280 and 260/230 should be greater than 1.9 and 1.8, respectively. If the 260/230 ratio is less than 1.8, it is advisable to perform RNA purification (using glycogen-sodium acetate precipitation method)⁸⁸.
• 30Reverse-transcribe the RNA into cDNA using Oligo(dT)20 primers. Performing RT-qPCR with Krt-14 primers will quantify the expression of Keratin-14 in all cell types⁵³ (Fig. 6a).

Fig. 6 |. Promoter specificity of plasmid construct for labeling and isolation of keratinocyte-derived exosomes.

a, Transcript abundance of Krt14 in primary mouse epidermal keratinocytes, fibroblasts, and macrophages (n=6). Data were shown as mean ± SEM and were analyzed by one-way ANOVA with the post-hoc Bonferroni’s multiple comparison test. b, The Exok-GFP were isolated from keratinocyte, fibroblast, and macrophage-conditioned media 48 h after transfection with lipofectamine, and NTA was done. The percentage of Exok-GFP from total particles in the conditioned media was plotted. c, Super-resolution confocal microscopic images showing expression of Exoκ-GFP in keratinocytes that are not visible in fibroblasts and macrophages. Scale, 20 μm. Figure is reprinted (adapted) with permission from Zhou et al., ACS Nano 2020, 14, 10, 12732–12748. Copyright (2020) American Chemical Society.

Reporter expression validation ● Timing 2–3 d

• 31Pipette 250 μl of the cell suspension (20,000 cells) of primary mouse keratinocytes, fibroblast, and endothelial cells to the microwell (0.70–0.75 mm deep) of collagen-coated glass bottom dishes (with No. 1.5 glass thickness) (n=3) and incubate the dishes at 37 °C in CO2 incubator. ▲CRITICAL STEP Collagen-coated dishes work well for primary cell culture. No. 1.5 coverslips are optimized to work best with all microscope objectives and for producing the highest quality images. Varying the thickness of the coverslip may lead to optical distortion and loss of resolution. ?TROUBLESHOOTING [AU: Should the Trobleshooting flag at Step 31 (and corresponding step in the Troubleshooting table) be changed to Step 44 – the step where the problem is detected? Please check and adjust accordingly if appropriate.]
• 32Allow cells to attach to the glass surface, and after 4 h, gently add 2 ml of cell culture growth medium, depending on the cell type to each dish. ▲CRITICAL STEP Filling the dish to the recommended level is critical to reducing the effect of evaporation and preventing osmolarity shifts.
• 33After 24 h, aspirate the medium and wash the cells thrice with 1 ml sterile cell culture PBS per dish.
• 34Aspirate PBS and add 2 ml exosome-depleted (Exo-D) medium in the cells.
• 35Transfect the cells with a cocktail of Krt14 promoter-driven plasmids encoding murine CD9, CD63, and CD81 with “in frame” GFP reporter (1 μg of each plasmid from Step 21 per dish) using Lipofectamine^™ 3000 Transfection Reagent as per the manufacturer’s instructions.
• 36Collect the conditioned media after 48 h for exosome isolation (Fig. 7) and perform NTA for the isolated exosomes to analyze the abundance of Exoκ-GFP53 (Fig. 6b).
• 37Wash the cells thrice with 1 ml sterile cell culture grade PBS and fix the cells using 4% (v/v) PFA for 15 min at RT. Wash the cells thrice with sterile cell culture PBS after fixation.

  ■PAUSE POINT Fixed cells can be stored at 4 °C in PBS up to 48 h and further processed later.

• 38Permeabilize the cells with 200 μl permeabilization buffer for 3 min.
• 39Wash the cells with PBS and block with 10% (v/v) normal goat serum (NGS) from host species of secondary antibody in PBS with 0.1% (v/v) Tween 20 (PBST) for 30–45 min at RT. CRITICAL STEP The use of blocking serum should be chosen based on the host in which the secondary antibody is raised. In the absence of appropriate serum, BSA can also be used to block. ▲CRITICAL STEP Addition of 0.1% Tween 20 reduces the surface tension and minimizes non-specific antibody interactions. ! CAUTION: Excessive blocking may result in less binding of antibodies.
• 40Remove the blocking buffer and incubate cells overnight at 4 °C with the anti-GFP primary antibody at a dilution of 1:200 in 2.5% (v/v) NGS in PBS with 0.1% (v/v) Tween 20 (PBST). ! CAUTION Do not wash the cells after blocking. ! CAUTION Longer incubation (>24 h) and a high antibody concentration can result in more background signals and overstaining of cells. ▲CRITICAL STEP It is important to include the appropriate controls (stained with cross-adsorbed antibody, only primary antibody and only secondary antibody) to validate the results. ▲CRITICAL STEP The working concentration of the antibody, the antibody diluent, and the incubation conditions such as time and temperature are the variables that might impact non-specific and antigen-antibody binding.
• 41Wash the cells thrice with 1 ml of 0.1% PBST (v/v) to remove the unreacted primary antibody and incubate with fluorescent secondary antibody at 1:200 dilution for 1 h at RT.

  ▲CRITICAL STEP Inadequate washing may lead to overstaining of the cells and non-specific background signals. The intervals between washes should be as short as possible to avoid drying out cells and creating artifacts from staining. ! CAUTION Longer incubation (>1 h) and a high antibody concentration can result in more background signals and overstaining of cells.

• 42Wash the cells thrice with 1 ml of 0.1% PBST (v/v) to remove the unreacted fluorescent secondary antibody and incubate in the appropriate concentration of DAPI (we use 1:10,000 dilution in PBS) for 8 min at RT. ! CAUTION: Longer incubation (>8 min) and a high DAPI concentration can result in cell overstaining.
• 43Wash the cells thrice with 1 ml of 0.1% PBST (v/v) to remove the unreacted DAPI and add fresh cell culture grade PBS/HBSS to each well. ■PAUSE POINT Stained cells can be stored at 4 °C up to 48 h in the dark and further processing for image acquisition later.
• 44Image the cells using a super-resolution confocal microscope (Fig. 6c).

Fig. 7 |. Schematic representation of the steps involved in testing promoter specificity of plasmid construct for labeling and isolation of keratinocyte-derived exosomes.

Schematic diagram showing exosome isolation process from murine keratinocytes, fibroblasts, and macrophages after transfection with a plasmid cocktail containing Krt14 promoter-driven recombinant plasmids encoding CD9, CD63, and CD81 with “in frame” GFP reporter to check the specificity of the plasmids for the keratinocytes. The figure was created with BioRender.com.

Tissue nanotransfection (TNT) on murine skin ● Timing 15 min

! CAUTION Any experiments involving animals should be undertaken only after obtaining institutional regulatory board permission.

• 45Anesthetize the mouse following the procedure approved by your Institution. We use continuous administration of 1–3% (v/v) of isoflurane.

  ?TROUBLESHOOTING

• 46Shave with hair trimmers the skin to be transfected and also put ear tags on the animals.

  ▲CRITICAL STEP Dehairing of the site at which TNT is to be applied must be undertaken 24–48 h prior to TNT.

• 47Apply depilatory cream to the shaved area for 30–60 s using a Q-tip.
• 48Remove the nairing cream first with a dry paper towel/cotton ball, followed by wet paper towels.
• 49Remove the mouse from anesthesia and allow it to regain consciousness before returning it to its cage.
• 50On the day of the TNT procedure (i.e. 24–48 h later nairing), anesthetize the animal following the procedure approved by your Institution.
• 51Wipe, clean the skin and then rub exfoliating cream in a circular motion on the area for 1 min.
• 52To remove the top dead layers of the epidermis and aid in cargo delivery penetration, tape strip the dehaired area at least 6 times with scotch tape.
• 53Clean the area with water and then alcohol wipes. ▲CRITICAL STEP Cleaning and eliminating the upper layers of the epidermis through tape stripping and exfoliating cream is important when trying to transfect cells in the dermis. Over-exfoliating should be avoided, as this can lead to the removal of deeper layers of the epidermis, resulting in a breach of skin barrier function.
• 54Insert a 26 G needle intradermally where the skin will be transfected (Fig. 8d).
• 55Using the hook clip wire, connect the 26 G needle to the positive terminal (red wire) of the pulse generator.
• 56Using a 1 ml pipette, add 250 μl of cargo (plasmids from Step 21) delivery solution (~100 ng/μl of each plasmid in PBS) to the reservoir of the nanofabricated chip ⁵⁰ (Fig. 8b).

  ▲CRITICAL STEP Appearance of liquid outside the reservoir indicates leakage between the reservoir and the PDMS seal. If leakage is observed, then that chip should not be used for TNT.

• 57Place the nanofabricated chip directly on the skin to cover the area from where 26 G needle is passing. Press the chip firmly by hand to ensure contact throughout the procedure (Fig. 8a, e, f) ▲CRITICAL STEP The chip must remain in contact with the skin throughout the electroporation process. This requires applying firm pressure (but not hard enough to break the nanoneedles of the chip) to avoid loss of contact due to any movement caused by the electric pulses, which vary by TNT location.
• 58Place the gold-plated electrode connected to the negative terminal (black wire) of the pulse generator such that the tip of the electrode is dipped in the plasmid solution in the reservoir on top of the nanofabricated chip. ! CAUTION The tip should be just dipped in the solution, and not touch the chip.
• 59
  Apply a square electric pulse (Fig. 8c). The TNT pulse generator parameters we use for epidermis are given in the following table:

  Parameter	Unit
  Driving pulse maximum current (PdA Limit):	300 mA
  Poration pulse voltage (Pp V):	100 V
  Poration pulse length (Pp on):	10 ms
  Poration interval (Pp off):	10 ms
  Driving pulse voltage (Pd V):	100 V*
  Driving pulse length (Pd on):	10 ms
  Driving Pulse interval (Pd off):	100 ms
  Number of pulses (Pd cycle):	10
  Decay rate:	0%
  Decay type:	Log

  ^*

  For transfecting cells in the dermis, this voltage needs to be between 150–200 V.

  Pulse Length: Duration of each pulse.

  Pulse Interval: Time between consecutive pulses. ▲CRITICAL STEP After turning on the power pack of the TNT power supply, ensure the pulse delivery in square wave.

• 60Check the Pd n list (Fig. 8g) to verify the successful delivery of the plasmid and carefully remove the nanofabricated chip and 26 G needle. ! CAUTION Mark the transfected area with India ink or with a permanent marker.
• 61Remove the mouse from anesthesia and allow it to regain consciousness before returning it to its cage.
• 62Clean the chip by removing the cargo solution and then dipping the chip in deionized water and then in 100% (v/v) ethanol and store it in an airtight plastic container at RT (15–30 °C) until its next use.

Fig. 8 |. In vivo delivery of plasmid construct for labeling and isolation of keratinocyte-derived exosome via Tissue nanotransfection.

a, Schematic representation of TNT on dorsal mouse skin. b, Image of plasmid in the reservoir of TNT chip. c, TNT pulse generator screen with the setup. d, Top view showing the electrode inserted intradermally on mouse dorsal skin. e, Top view showing the placement of TNT chip and positive electrodes. f, Side view showing the placement of TNT chip and electrodes for the successful delivery of plasmid. g, Pd n list to check whether the delivery is successful or not.

Wounding procedure on animal. ● Timing 20 min

• 63Next day, anesthetize the mouse following the procedure approved by your Institution. We use continuous administration of 1–3% (v/v) of isoflurane.
• 64Clean the shaved TNT-transfected area with water and then an alcohol wipe.
• 65Create four full-thickness excisional wounds of 8 mm diameter, equidistant from the midline, on the dorsal skin of mice with a 8 mm disposable biopsy punch and collect the skin (day 0; d0) tissue in microcentrifuge tubes, formalin, and OCT casettes for further experiments.
• 66Bandage the mouse and then remove the mouse from anesthesia and allow it to regain consciousness before returning it to its cage. ▲CRITICAL STEP Administration of analgesics to relieve the pain as per IACUC protocol (23083167).

Euthanizing animals and harvesting skin tissue for isolation of endosomal-originated exosomes of keratinocyte origin. ● Timing 30 min

• 67Euthanize the animal according to your institutional animal regulatory board guidelines at an appropriate time after the TNT application. ▲CRITICAL STEP After euthanasia, tissue must be harvested immediately to avoid dehydration. Dehydration processes may impact tissue, which might be detrimental to the molecular properties and quality of exosomes. Any procedure that might cause necrosis or apoptosis needs to be avoided.
• 68Collect the WE tissue samples using 8–12 mm biopsy punches from the region where TNT was performed after 24–48 h. ▲CRITICAL STEP Wound-edge tissue samples must be harvested with a biopsy punch that encompasses the WE with a rim of <2 mm of unwounded tissue to avoid the exosomes from surrounding healthy tissue. ▲CRITICAL STEP Once harvested, the samples must be processed immediately. ■PAUSE POINT However, if a pause is needed, which should be avoided, if possible, then it is recommended to store these samples in liquid nitrogen or −80 °C. Storing samples at −80 °C will slow down enzymatic processes that might cause tissue deterioration.
• 69To visualize the Exo_κ-GFP within tissue, collect samples using biopsy punches in microcentrifuge tubes in OCT compound. ■PAUSE POINT The samples in OCT can be stored at −80 °C and later used for histological, immunohistochemical, and molecular studies.

Immunohistochemistry ● Timing 2 d

• 70Dissect tissue from step 69 and place it into cryomolds with OCT.
• 71Immediately flash freezes on an aluminum foil boat floating on liquid nitrogen.
• 72Cryosection tissue (10 μm thick sections) using Cryostar NX50 on glass slides.
• 73Wash with PBS to remove OCT and then fix in ice-cold acetone for 5 min.
• 74Wash sections in PBS for 3 min (3–5 dips) followed by 30–45 min of blocking in 10% (v/v) NGS.

  CRITICAL STEP If you are working with mouse tissue samples and using a primary antibody raised in mouse, then you need to use 10% (v/v) MOM along with 10% (v/v) NGS for blocking endogenous mouse immunoglobulins.

• 75Incubate tissue overnight at 4 °C in an appropriate concentration of primary antibody in 2.5% (v/v) NGS in PBS with 0.1% (v/v) Tween 20. We used anti-GFP antibody at a dilution of 1:200.
• 76Wash tissue thrice in PBS-T (5 min per wash) and incubate with an appropriate concentration of fluorescent secondary antibody for 1 h at RT. To detect GFP, we used goat anti-mouse IgG Alexa Fluor^® 405 antibody, goat anti-chicken IgG Alexa Fluor^® 488 antibody, and goat anti-rabbit IgG Alexa Fluor^® 568 antibody at 1:200 dilutions (Table 4).
• 77Wash tissue thrice in PBST (5 min each), then incubate in an appropriate concentration of DAPI (we use 1:10,000 dilution in PBS) for 8 min.
• 78Wash samples thrice in PBST (5 min each), put mounting media, and mount coverslips.
• 79Allow the stained slides to dry overnight, clean the slides the next day, and do imaging.

  For the images shown in Fig. 9a, b we used a laser scanning confocal microscope and quantitatively estimated the GFP expression using Zen Blue 3.8 software (Fig. 9c). Similarly, exosomes from macrophages and fibroblasts can be labelled using this technique by selecting an appropriate promoter and reporter (Fig. 3a, b).

Fig. 9 |. Visualization and abundance of keratinocyte derived exosomes in murine WE tissue.

a, Confocal microscopic images showing the presence of Exoκ-GFP in the dermis. The white dashed line shows the epidermal–dermal junction. Scale, 10 μm b, Super-resolution airyscan confocal microscopic images showing GFP-labeled exosomes (0.2 μm is the depicted size of exosomes using scale bar) in the dermis. Exoκ-GFP are indicated by red arrowheads in a and b. Images were obtained 24 h post TNT with Krt14 promoter-driven plasmids. Scale, 2μm c, Quantification of Exok-GFP in epidermis and dermis at 6, 12, 24, and 48 h post-TNT with Krt14 promoter-driven recombinant plasmids. Data in c are shown as mean±SEM and were analyzed by one-way ANOVA with the post-hoc Bonferroni’s multiple comparison test. Blue and red dots represent the biological replicates, while grey dots represent the technical replicates for Exok-GFP abundance in epidermis and dermis. The entire figure is reprinted with permission from Zhou et al., ACS Nano 2020, 14, 10, 12732– 12748. Copyright (2020) American Chemical Society.

Isolation of cell type-specific exosomes ● Timing 2 d

• 80Take the samples from step 69 out from liquid nitrogen storage into a pre-filled liquid nitrogen dewar.
• 81Use pre-cleaned tweezers to transfer the tissue from the liquid nitrogen dewar to a pre-weighed 1.5 ml microcentrifuge tube ! CAUTION The cap of the 1.5 ml microcentrifuge tube should be poked with an 18 G needle to avoid popping up from the residual liquid nitrogen.
• 82Weigh the tissue with the pre-weighed 1.5 ml microcentrifuge tube. ▲CRITICAL STEP Every time after weighing, the operator needs to put the samples back in liquid nitrogen to avoid the onset of lipase and protease activity that might affect the yield of the exosomes.
• 83Pulverize the tissue sample in liquid nitrogen with a pestle and mortar until it becomes powder. ▲CRITICAL STEP Do not thaw the tissue sample on ice before homogenization. ▲CRITICAL STEP Clean the pestle and mortar after homogenizing every sample with a thick paper towel to avoid contamination.
• 84Transfer the pulverized tissue to 1 ml microcentrifuge tube and add 500 μl of sterile PBS to it. After this, vortex the dispersed solution in a vortex mixer for one minute and then centrifuge for 30–45 s at 2000 × g ▲CRITICAL STEP The weight of the tissue determines the volume of PBS needed to resuspend it. For every 100 mg of tissue add 500 μl of sterile PBS.
• 85Transfer the supernatant to a fresh 1.5 ml microcentrifuge tube.
• 86Add 500 μl of PBS again to the tissue pellet in the microcentrifuge tube.
• 87Vortex the dispersed solution in a vortex mixer for one minute and centrifuge for 30–45 s at 2000 × g to separate the supernatant.
• 88Again, take out the supernatant and add it to the tube in step 85.
• 89Centrifuge approximately 1 ml of tissue extracted solution for 15 min at 5,000 × g in a cold centrifuge and then collect the supernatant in a fresh 1.5 ml microcentrifuge tube.

  ▲CRITICAL STEP It is mandatory to carefully balance the samples to avoid unbalancing before placing the tubes in the centrifuge. For balancing, add PBS to make the same volume in all the tubes.

• 90Discard the tissue pellet.
• 91Centrifuge collected supernatant at 45 min at 10,000 × g in a cold centrifuge.
• 92Transfer the supernatant to a new tube and discard the pellet.
• 93Meanwhile, wash the anti-GFP beads (30 μl per sample) with 1 ml dilution buffer thrice and separate the beads using magnet. ▲CRITICAL STEP Washing of GFP beads with dilution buffer is very critical as the bead slurry contains 20% (v/v) ethanol which may interfere with the binding of exosomes and exosome functionality. ▲CRITICAL STEP Amount of GFP beads slurry required per sample depends on the type and size of the sample. In general, 25 μl of GFP bead slurry can bind up to 15–20 μg of recombinant GFP as per the manufacturer’s instructions.
• 94Block the washed anti-GFP beads by incubating with 1 ml blocking buffer. Resuspend the GFP beads by gently inverting the tube or pipetting up or down.! CAUTION Do not vortex the beads! Vortexing can cause beads to adhere to the tube walls, making them difficult to resuspend even after centrifugation. Additionally, the mechanical forces generated during vortexing may disrupt complexes formed during the mixing process, compromising the efficiency of target molecule capture. In most protocols, gentle mixing or pipetting is recommended to resuspend beads, as these methods minimize bead loss and maintain the integrity of the complexes.
• 95Keep the GFP beads with blocking buffer in rotation on a tumble rotator in a cold room for 30–45 min. ! CAUTION Longer incubation leads to excess blocking which may result in less exosome capture.
• 96Separate the GFP beads with a magnet until the supernatant is clear.
• 97Discard the supernatant. ! CAUTION Do not wash the beads after blocking!
• 98Add the supernatant from the tissue extract from step 92 to the equilibrated GFP beads.

  ▲CRITICAL STEP 30 μl of GFP beads are required for 150–200 mg of tissue sample. If you have a tissue sample weighing more than 200 mg, add more GFP beads accordingly.

• 99Rotate these tubes end-over-end on a tumble rotator in a cold room overnight to bind the GFP-labeled keratinocyte origin exosomes (Exo_κ-GFP) to the anti-GFP beads.
• 100Next day, separate the Exo_κ-GFP conjugated with anti-GFP beads from the dispersion. First, centrifuge the eppendorf tubes at 2500 × g for 3 min and then, with the help of a magnet attract the beads and carefully take out the supernatant. ▲CRITICAL STEP Supernatant contains the exosomes from other cell types except keratinocytes. Always store approximately 500 μl of supernatant (flow through or non-bounded exosome fraction) in −80 °C for further analysis.
• 101Resuspend the Exo_κ-GFP containing beads in 500 μl of wash buffer/dilution buffer.

  ▲CRITICAL STEP Washing with dilution buffer is important because proteins may be adsorbed on the bead surface in a non-specific manner, leading to false interpretation.

• 102Remove the wash buffer/dilution buffer from the Exo_κ-GFP conjugated with anti-GFP beads. ▲CRITICAL STEP For flow cytometry of beads, do not elute the exosomes from the anti-GFP beads and add 500 μl PBS to the Exo_κ-GFP conjugated with anti-GFP beads. From this step, directly proceed to step 155. For characterization of the Exo_κ-GFP using NTA, western blot, ELISA, electron microscopy, or functional assays, proceed to step 108.
• 103Wash the GFP-Trap beads three times in PBS. Then, for elution of intact beads, add 50 μl of glycine (0.2 M, pH 3) and constantly pipette up and down for 30–45 s at room temperature. ! CAUTION Do not vortex the beads! ▲CRITICAL STEP Acidic pH results in a high yield of exosomes by disrupting antigen-antibody interactions.

  ▲CRITICAL STEP Elution at RT is more efficient than elution at 4 °C.

• 104Separate the Exo_κ-GFP containing GFP beads with a magnet until the supernatant is clear.
• 105Transfer the supernatant (containing Exo_κ-GFP) to a new 1.5 ml microcentrifuge tube and immediately add 10 μl of 1.0 M Tris pH 8.0 (neutralization buffer) to adjust the pH to 7.4. ▲CRITICAL STEP Regaining neutral pH after elution of exosomes from the beads is extremely important as exosomes can lose some stability when left at pH <3 for an extended period.
• 106Repeat the elution (Step 103–105) one more time to increase the elution efficiency.
• 107Pool the eluted Exo_κ-GFP and add to a clear 1.5 ml ultracentrifuge tube, adding PBS to make the volume up to 1 ml. Ultracentrifuge the samples at 245,000 × g for 1.5 h at 4 °C. Remove and discard the supernatant and then collect the Exo_κ-GFP from the bottom of the tube very carefully (approx. 130–140 μl). ▲CRITICAL STEP Ensure that the tubes are balanced for ultracentrifugation for safe operation. ▲CRITICAL STEP It is recommended to use fresh exosome aliquots in PBS for NTA, TEM, and SEM. For performing BCA, proteomics, transcriptomics, lipidomics, and RNA analysis, either fresh or frozen aliquots may be used. ■PAUSE POINT Exosome samples can be stored at 4 °C for a week, but −80 °C is preferable for long-term storage for further analysis.

Characterization of exosomes

Nanoparticle tracking analysis (NTA) ● Timing 1 h for 4 samples

• 108Turn on the NanoSight Pro instrument and start the NS Xplorer software on the desktop computer.
• 109Using a 1 ml tuberculin syringe, inject 10 ml of 0.2 μm filtered UPW into the cell channel through the inlet port to remove any impurities present in the flow cell.

  ▲CRITICAL STEP Proper cleaning of the flow cell increases the reading accuracy because of less scattering of light by the NTA machine. ! CAUTION Make sure the outlet port is connected to the screwcap bottle to collect the waste solution. Immediately close the inlet port after injection.

  ?TROUBLESHOOTING

• 110Repeat step 109 if you still observe particles in the live-view screen of the software.

  ▲CRITICAL STEP This step should be performed until all particles are removed from the flow cell.

• 111
  Dilute the standards in UPW according to the table below:

  Beads size	Dilution factor	Beads volume (μl)	UPW (μl)
  100 nm	500	4	1996
  200 nm	55	36	1964

• 112Inject the diluted standards using a 1 ml syringe into the analyzer.
• 113Initiate auto-alignment and auto-focus performance. The initial flow rate is set to

  1.5 μl/min for 3 s and then changes according to the parameters set by the user. The syringe pump can support flow rates in the range of 0.1 to 200 μl/min.

  The parameters given to the instruments are adjusted manually:

  Properties	Parameters
  Distribution type	Finite Track Length Adjustment (FTLA)
  Flow rate	Static
  Display brightness	2–5

• 114Wash the beads out of the instrument by flushing the cell channel with UPW until you see particles moving on the screen.
• 115Dilute the exosomes samples in UPW prior to injection. ▲CRITICAL STEP The dilution depends on the yield of exosomes required. Optimized reading results fall under the range of 100–400 particles per field view.
• 116Inject diluted exosomes and measure the size and concentration of the exosomes(Fig. 4a).
• 117Set the brightness to a value of 2–5 for the exosome measurements. Adjust brightness to filter out strong light-scattering particles or to amplify weakly scattering artifacts as needed. For exosome analysis, the flow rate is maintained at 4 μl/min for the duration of the experiment.
• 118Acquire the video and perform 5–10 technical replicates of each sample to avoid instrumental errors. ▲CRITICAL STEP It is also mandatory to do deep cleaning of the chamber if the instrument error is not cleared by repeatedly washing with the detergent and ethanol provided by the manufacturer. ! CAUTION Particles that scatter too much light may overlap and be excluded from the analysis by mistake. ! CAUTION It is important to protect the laser module from damage by not pouring any liquid on it.
• 119Flush the cell channel with the UPW between experiments. ▲CRITICAL STEP Ultra-pure distilled water is used instead of PBS as PBS contains crystals that can scatter more light, resulting in false interpretations. It is mandatory to flush UPW between experiments so that the results are devoid of impedence by the earlier experiments.
• 120Wash the cell with filtered UPW and inject the next sample and repeat steps 115– 119.

Zeta potential ● Timing 10 min per sample

• 121Disperse 20 μl of isolated exosomes (10⁹ particles/ml as obtained from NTA) in 1 ml of UPW with the help of a pipette. ▲CRITICAL STEP The surface charge and surface chemistry of exosomes can change depending on their state of dispersion. As the PBS concentration rises, the exosomes’ negative surface charges decrease. This effect is caused by the presence of more counterions, leading to increased conductivity, which can hold the electrical double layer (EDL) more firmly and cause the EDL to shrink.
• 122Load the exosome suspension in a Zetasizer instrument cell, and insert the cell into the holder with the correct orientation. Press down until the cell clicks into place and measure the surface charges in volume-weight size distribution mode, following the manufacturer’s instructions.

Transmission electron microscopy ● Timing 1 h per sample

• 123Fix the Exo_κ-GFP (1–5 μl, 10⁸ exosomes) in a solution containing 10 μl of 0.2 M of sodium cacodylate buffer, 2 μl of 16% (w/v) of an aqueous solution of PFA, and UPW to make a final volume of 20 μl. The resulting solution will be 0.1 M of sodium cacodylate and 2% (v/v) PFA. ! CAUTION Both sodium cacodylate and PFA are toxic compounds. Avoid inhalation, ingestion, and skin contact. Perform cell fixation in a hood. Wear gloves, masks and PPE kit.▲CRITICAL STEP It is imperative to be mindful of the amount of exosomes to be fixed. 1 μl of 30% (v/v) sucrose fraction is commonly added to the fixed exosomes for clear visualization. ■PAUSE POINT At this stage, the fixed exosomes may be stored at 4 °C for several days.
• 124Position the 300 μm carbon-coated copper grid in the TEM grid holder.
• 125Expose the grid to a glow discharge of 20 mA for 30 s at 0.39 mbar. ▲CRITICAL STEP Using PELCO easiGlow automated glow cleaning system is recommended to minimize variability between samples.
• 126The carbon-coated grid must point upward. To achieve this, the grid must be held with self-clamping tweezers. Gently drop 2 μl of fixed exosomes on the discharged grid and incubate for 30 s to allow for adsorption of exosomes to the grid.
• 127Absorb the excess solution with Whatman filter paper. Add 1% (w/v) uranyl acetate to negatively stain the exosomes, incubate for 30 s and immediately remove the stain with filter paper. ! CAUTION Uranyl acetate is a toxic solution. Avoid inhalation, ingestion, and skin contact and use hood while performing this step. ▲CRITICAL STEP It is essential to avoid overstaining the exosome samples, to clearly be able to visualize the exosomes.
• 128Let the grid air dry overnight.
• 129Image exosomes via JOEL JEM 1400plus transmission electron microscope equipped with a 4000 × 4000-pixel Gatan CCD camera.

Exosome antibody array assay ● Timing 18 h

• 130Take an exosome sample resuspended in 880 μl of 1X DPBS. ! CAUTION Keep exosomes on ice as heat may change their composition. Phosphate buffered saline preserves the structure and composition of exosomes and stabilizes them.
• 131Isolate proteins from exosomes. Add 100 μl of 1X Cell lysis buffer which should be prepared by diluting 10X cell lysis buffer (see reagent preparation) in UPW and adding 10 μl of 1X PIC and 10 μl of 1X PMSF immediately before use. ■PAUSE POINT At this point, the lysates may be stored in 4 °C for short term storage (same day use), and −80 °C for long term storage. When storing in −80 °C it is recommended to aliquot the lysates to mitigate the effects of freeze thaw cycles. The lysates may also be stored at −20 °C in 50% (v/v) glycerol. The lysates can be stored for up to a year and then discarded.
• 132Perform BCA analysis with two technical replicates in a 96 well plate, following the manufacturer’s instructions.
• 133Measure the absorbance at 562 nm on a plate reader. ! CAUTION 562 nm is essential because it is the wavelength that detects the purple complex that proteins form with the working reagent. ▲CRITICAL STEP It is important to perform blank reduction.
• 134Prepare a standard curve by plotting the average blank-corrected 562 nm measurement for each BSA standard vs. its concentration in μg/ml.
• 135Determine the protein concentration of each unknown sample from the average of the replicates. ! CAUTION Do not proceed if the R value is less than 0.9.
• 136Add cell lysis buffer to 50 μg of sample from Step 131 for a final concentration of 1X Cell lysis buffer. ! CAUTION The protocol is optimized for 50 μg protein. If you have less than 50 μg, it is advised that you combine one or more samples to make the total protein amount 50 μg.
• 137Vortex for 15–30 s. ▲CRITICAL STEP It is essential that the mixture is vortexed well so the lysis buffer may interact with the exosomes to break up their membranes to liberate the protein contents inside the exosomes and in their membranes.
• 138Add 1 μl of labeling reagent brought to RT to the lysate.
• 139Incubate this mixture at RT for 30 min with constant agitation on a shaking rotator.

  ▲CRITICAL STEP Constant agitation is essential to aid the labeling reagent in interacting with the proteins of interest and binding with them. ! CAUTION 30 min is the optimal amount of time for the labeling reagent to react with the proteins. Less time would be insufficient to bind all relevant proteins while more than 30 min would yield nonspecific binding. Remove excess labeling agent using a filter column. ! CAUTION Excess labeling agent may lead to non-specific binding. Vortex the column to resuspend the medium.

• 140Loosen the cap.
• 141Twist off the bottom.
• 142Place the column in a collection tube and centrifuge (800 × g at RT for 1 min) for the removal of storage buffer.
• 143Discard the storage buffer. ▲CRITICAL STEP It is important to properly discard the storage buffer to avoid unwanted contaminants, which may affect downstream protein detection. ! CAUTION The storage buffer may interfere with the calibration of the column and the elution of the proteins of interest.
• 144Equilibrate the column with 400 μl of column buffer. ▲CRITICAL STEP It is important to follow the manufacturer’s protocol as specified, as the ingredients of reagents are proprietary information that hasn’t been disclosed. Therefore, exactly 400 μl of column buffer must be added.
• 145Centrifuge at 800 × g for 1 min and discard the flow through. Repeat this 5 times.

  ▲CRITICAL STEP Calibrating the column this way is important to ensure proper purification of the sample.

• 146Apply 100–180 μl of the sample to this column and elute by centrifuging 800 × g for 2 min at RT. ▲CRITICAL STEP It is imperative to apply the sample directly in the middle of the column to ensure all the sample is filtered out. ! CAUTION If the sample is less than 140 μl, add a stacker volume of column buffer to reach 140 μl.
• 147Combine the eluent with 5 ml blocking buffer and homogenize by inverting 2–3 times. ▲CRITICAL STEP It is essential to block the sample to mitigate nonspecific binding.
• 148Apply the mixture to the membrane (face up) that was previously wet in distilled water for 2 min. ▲CRITICAL STEP It is imperative to apply the sample to the binding side of the membrane, or else protein expression will be undetectable.
• 149Write your initials and the date on the top right-hand corner so one can tell which side is up. ▲CRITICAL STEP Handle the membrane carefully with flat-toothed forceps. If handled with other forceps, the membrane may fold or tear.
• 150Incubate the membrane at 2–8 °C overnight (~16 h) on a standard analog rocker at 30 rpm. ! CAUTION If the membrane is incubated at RT, it should not exceed an incubation period of more than 2 h to maintain the integrity of the proteins bound by the membrane.
• 151Once the incubation is complete, decant the blocking buffer. Add 5 ml 1X wash buffer and incubate with agitation for 5 min at RT. Repeat the wash step twice and decant the wash buffer.
• 152Make the detection buffer mixture with 5 ml of detection reagent A and 2.5 μl of detection reagent B. Add 5 ml of the detection buffer mixture to the membrane and incubate for 30–45 min at RT with constant agitation. ▲CRITICAL STEP Keeping the membrane in the detection buffer for enough time is important so the HRP-conjugated detection buffer can bind to proteins of interest. Agitation is important to help HRP interact with the proteins. ! CAUTION One vial of the detection buffer must be used per experiment as advised by the manufacturer.
• 153Decant the detection buffer, add 1X wash buffer, and incubate at RT for 5 min with agitation. Wash the membrane three times. ▲CRITICAL STEP It is imperative to wash the membrane well to remove excess detection buffer so the developing mixture can interact with the membrane efficiently. ! CAUTION It is important to keep the wash buffer and detection buffer at RT because these buffers are stable at RT and do not need to be heated. Heating the buffers may denature antibodies and if the buffers are too cold it may cause -protein shock potentially damaging their structure and function ■PAUSE POINT If you are not ready to develop the membrane right away, it may be stored in a box in PBST at 4 °C.
• 154Spray ECL on the membrane and incubate it for 5–6 min at RT then image immediately. !

  CAUTION The various antibody spots will provide signals of varying degrees of intensity depending on the source of exosome isolation. For a given sample, signals from each spot on the array cannot be directly compared since the antibody concentrations are different across the array. However, signals can be compared across samples for the same spot to provide a qualitative assessment of the intensity of the spots visualized (Fig. 4b)

  ?TROUBLESHOOTING

Flow cytometry ● Timing 2 h from incubation with antibody to analysis

▲CRITICAL STEP GFP-Trap Magnetic Agarose beads are used for FACS analysis, as exosomes (<150 nm) are too small to visualize by FACS. By using these beads which are 40 μm in diameter, flow cytometry can be performed with the assumption that multiple exosomes are bound to each bead, and the overall size of particles then substantially exceeds the lower limit for FACS detection, which is 500 nm.

• 155Stain the beads conjugated with exosomes with fluorescein-conjugated antibodies for exosomal membrane markers (Rab 7, and HSP90)^89,90 in 500 μl volume of PBS buffer at the antibody concentration recommended by the manufacturer. ▲CRITICAL STEP Always stain beads (without exosomes) with fluorescein-conjugated antibodies as a negative control. Additionally, unstained beads with exosomes should always be used as a control along with stained beads. For Rigor, other exosomal markers (LAMP1, Caveolin1 and Rab5) can also be studied.
• 156Incubate at RT for 30 min with rotation.
• 157After staining, wash the beads thrice with 500 μl PBS buffer and centrifuge at 2500 × g for 3 min and then with the help of magnet attract the beads and discard the supernatant.
• 158Resuspend the stained beads in 500 μl of PBS buffer, then use the appropriate isotype control-stained beads to analyze them using flow cytometry with a medium flow rate of 35 μl/min with 5000–10000 events (Fig. 4c).

  ?TROUBLESHOOTING

Sucrose density gradient ultracentrifugation of exosomes ● Timing 14 h

• 159
  Prepare 0.5 M, 1 M, 1.5 M, and 2.5 M sucrose solutions in 1 mM PBS.

  Molarity of Sucrose	Density (g/cm3)
  0.5 M	1.06
  1 M	1.12
  1.5 M	1.19
  2.5 M	1.31

• 160In a 1.5 ml clear ultracentrifuge tube, carefully layer 250 μl of 2.5 M sucrose solution, followed by 1.5 M, 1 M and 0.5M.
• 161Resuspend the exosome pellet after eluting from beads (approximately 10¹⁰ particles/ml) in 250 μl of 0.5 M sucrose solution by pipetting up and down. ▲CRITICAL STEP Ensure that exosomes are not stuck to the tube in clumps and are in fact resuspended in the solution.
• 162To the ultracentrifuge tube from Step 160, add 0.5 M sucrose equilibrated exosomes, carefully layering them on top of the gradient. ! CAUTION Make sure to add the solution by the edge of the tube.
• 163Then centrifuge for 100,000 × g for 12 h at 4 °C. ! CAUTION Mishandling of an ultracentrifuge is hazardous. All 1.5 ml ultracentrifuge tubes should be filled ¾ with no imbalance. Therefore, it is recommended to weigh the tubes before loading them into the ultracentrifuge. ▲CRITICAL STEP Ensure that the tubes are balanced by weighing them. If the weight difference between tubes is drastic (>0.01 g) then balance with one or two drops of 0.5 M sucrose solution. A balance tube may be used in case of an odd number of samples. The balance tube must have the same sucrose gradients as the sample tubes. Other solutions like PBS, water or other solvents should not be used to balance out the samples with sucrose solutions. ▲CRITICAL STEP Ensure minimal disturbance to the samples while loading the centrifuge. It is recommended to use a low-braking setting at the end of the centrifugation.
• 164The exosomes appear as white colored ring in the second layer of sucrose (1 M-1.5 M), having a density in the range of 1.12–1.19 g/cm³. ▲CRITICAL STEP If you do not observe the white ring, then repeat the ultracentrifugation with a new gradient, loading a higher concentration of exosomes.
• 165To pellet down these exosomes for further use, discard the first 250 μl from the top of the tube and then collect the exosomes from the second layer (250 μl) of the tube in a new ultracentrifuge tube.
• 166Add 850 μl of ice-cold PBS and mix by pipetting. ! CAUTION Do not vortex!

  ▲CRITICAL STEP Owing to the density and viscosity of the medium when sucrose molarity is high, the addition of extra PBS will lower the density and viscosity of the solution and facilitate pelleting.

• 167Weigh the tubes to ensure they are balanced and centrifuge at 100,000 × g for 70 min at 4 °C.
• 168Discard the supernatant (approx. 850 μl). Collect the 150 μl from the bottom of tube which contains exosomes and make smaller aliquots of 50 μl without adding any buffer or reagent. ! CAUTION It is not recommended to freeze thaw the exosome solution several times. ▲CRITICAL STEP It is recommended to use fresh exosome aliquots in PBS for NTA, TEM, and SEM. While performing BCA, proteomics, transcriptomics, lipidomics, and RNA analysis either fresh or frozen aliquots may be used. ■PAUSE POINT The aliquots may be stored at −80 °C for several months.

Troubleshooting

Troubleshooting suggestions are shown in Table 5.

Timing

Steps 1–15, Transformation of plasmid DNA in E. coli DH5α competent cells: 2 d Steps 16–21, Plasmid amplification and isolation: 20 h

Steps 22–30, Promoter validation: 30 h

Steps 31–44, Reporter expression validation: 2–3 d

Steps 45–62, Tissue nanotransfection (TNT) on mouse skin: 15 min Steps 63–66, Wounding procedure on mice: 20 min

Step 67–69, Euthanizing animals and harvesting skin tissue for isolation of endosomal-originated exosomes of keratinocyte origin: 30 min

Step 70–79, Immunocytochemistry: 2 d

Step 80–107 Isolation of cell-specific exosomes: 2 d

Step 108–120, Nanoparticle Tracking Analysis (NTA): 1 h for 4 samples Step 121–122, Zeta Potential: 10 min per sample

Step 123–129, Transmission electron microscopy (TEM): 1 h per sample Step 130–154, Exosome antibody array assay: 18 h

Step 155–158, Flow cytometry: 2 h from incubation with antibody to analysis Step 158–168, Sucrose density gradient: 14 h

Anticipated results

We have provided a comprehensive protocol for in vivo labeling and isolating cell type-specific exosomes, from a heterogeneous pool of EVs. We discussed the steps involved in the (a) transformation, amplification, and isolation of plasmids to deliver them via electroporation, (b) application of the TNT technique, and (c) isolation and characterization of cell type-specific exosomes. The three tetraspanins CD9, CD63, and CD81 are reliable markers of exosomes as per EVPedia and Exocarta.⁹¹ We designed three plasmids encoding murine keratin 14 (Krt14) promoter-driven CD9, CD63, and CD81 with an “in frame” GFP-reporter (Fig. 1a) to allow the expression of GFP-tagged CD markers only in keratinocytes. We have validated the specificity of the plasmids in primary murine cells, but murine cell lines can also be used to validate keratinocyte-specific expression. The delivery of plasmids encoding K14 promoter-driven CD9, CD63, and CD81 GFP fusions plasmids via TNT was proven by super-resolution confocal microscopy which demonstrates the presence of exosomes with GFP-reporter expression in the epidermis and dermis (Fig. 9a, b). Pure keratinocyte-derived exosomes were isolated from WE tissue by affinity selection using GFP magnetic beads, followed by separation and purification of exosomes using ultracentrifugation. We characterized the cell type-specific exosome population isolated from tissues in depth to validate our approach. The NTA characterization reveals the average size of exosomes around 105 nm in Fig. 4a. With the help of EM, the size and morphology of exosomes can be revealed as shown in Fig. 2b. Raman spectroscopy analysis showed that differential ultracentrifugation and magnetic bead separation can separate small EVs from large EVs.⁸⁴ Enriched proteins such as TSG101, ALIX, FLOT1, ANXA5, and GM130 also validate the successful isolation of small vesicles like exosomes as shown in Fig. 4b. Flow cytometric analysis of GFP-trap beads post-adsorption with Exoκ‑GFP following incubation with fluorescently tagged antibodies targeting exosome markers Rab7A and HSP90 showed an increase in fluorescence intensity as compared to unstained exosomes with beads (Fig. 4c), demonstrating the successful isolation of Exoκ‑GFP from murine tissue homogenate.

Table 1.

Overview of the Procedure

Stage	Objective	Procedure
Plasmid Design (Experimental Design)	Ensure accurate and reproducible labeling of cell type-specific exosomes in vivo.	○ Design and construct plasmids with the required cell specificity. ○ Outsource plasmid synthesis to a reliable manufacturer, such as Applied Biological Materials (ABM) Inc.
Plasmid Transformation (Steps 1 – 15)	Amplify the synthesized plasmid and store it for future use.	○ Transform the plasmids into competent DH5α cells. ○ Store the transformed cells in a glycerol stock solution (25% final concentration) at −80 °C.
Plasmid Amplification and Isolation (Steps 16–21)	Isolate the recombinant plasmid DNA.	○ Amplify the plasmids by culturing the transformed DH5α cells overnight at 37 °C. ○ Isolate the plasmids using a commercially available plasmid purification kit mentioned in the Reagents section, following the manufacturer’s protocol.
Plasmid Validation (Steps 22–44)	Confirm the specificity of promoters and the expression of reporters.	○ Validate the plasmids in vitro using primary murine cells before in vivo delivery.
Plasmid Delivery (Steps 45–62)	Deliver the validated plasmids into target tissues.	○ Use topical TNT or a in vivo fectamine-based approach for plasmid delivery. ○ Tissue nanotransfection technique is preferred to control the depth of plasmid delivery using voltage adjustments.
Wounding procedure (Steps 63–66)	Create excisional wounds on the dorsal skin of mice	○ Perform excisional wounding with the help of biopsy punches.
Harvesting tissue (Steps 67–69)	Collect the targeted murine tissue post-euthanasia.	○ Collect the WE tissue where the plasmid was delivered. ○ Ensure the collection is within 2 mm from the WE.
Immunohistochemistry (IHC) (Steps 70–79)	Verify reporter expression post-plasmid delivery.	○ Perform immunohistochemistry on serial sections of the transfected WE tissue. ○ Use either formalin-fixed or Optimal Cutting Temperature (OCT) embedded tissue samples.
Isolation of Cell type-Specific Exosomes (Steps 80–107)	Isolate exosomes originating from specific cell types.	○ Confirm reporter expression within the tissue. ○ Isolate keratinocyte-derived exosomes using differential centrifugation. ○ Follow the immunomagnetic separation using magnetic agarose beads conjugated with reporter antibody. ○ Using acidic elution method, elute the exosomes and further purify them using ultracentrifugation.
Characterizations (Steps 108–168)	Characterize the isolated exosomes.	○ Determine size and concentration using NTA. ○ Measure surface charge using zeta potential. ○ Assess morphology using SEM and TEM. ○ Evaluate density, presence of markers, and absence of contaminants using Exosome Antibody Array assay and Flow Cytometry.

Table 2 |.

Plasmid details

	CD9	CD63	CD81
Product name	pLenti- Krt14-CD9-GFP Vector	pLenti- Krt14-CD63-GFP Vector	pLenti- Krt14-CD81-GFP Vector
Catalogue number	LV437001-Custom	LV485877-Custom	LV457581-Custom
Lot number	0027844751006	0027844751002	0027844751004
Storage conditions	−20 °C	−20 °C	−20 °C
Insert site	Insert CD9 in frame between Lys152 of Krt14 and Met1 of GFP	Insert CD63 in frame between Lys152 of Krt14 and Met1 of GFP	Insert CD81 in frame between Lys152 of Krt14 and Met1 of GFP
CDS of accession number	NM_007657.4 without stop codon	NM_001042580.1 without stop codon	NM_133655.2 without stop codon
Size	9821 bp	9857 bp	9851 bp
Cloning site	NheI/EcoRV (Seamless cloning)	NheI/EcoRV (Seamless cloning)	NheI/EcoRV (Seamless cloning)
Promoter	Krt14	Krt14	Krt14
Reporter	GFP	GFP	GFP
Selection marker in bacteria	Kanamycin	Kanamycin	Kanamycin
Selection marker in mammalian cells	Puromycin	Puromycin	Puromycin
Insert name	CD9	CD63	CD81
Insert size	678 bp	714 bp	708 bp
Competent cell	DH5α	DH5α	DH5α

Table 3 |.

Proteins targeted by antibodies used in the protocol and their relevance

Antibody	Relevance in exosome detection
CD 63	CD stands for cluster differentiation.92 CD63 is a tetraspanin that is present in the exosome membrane.
EpCAM	It stands for Epithelial Cell Adhesion Molecule and is a transmembrane protein, which is also an exosome biomarker.93
ANXA5	Annexin A5 is another known exosome protein marker, that acts as exosomal cargo and binds to phosphatidylserine (PS) which is found in the membrane of apoptotic cells.93
TSG101	This is known as the Tumor Susceptibility Gene 101 which is a protein that is a part of ESCRT-1 complex and aids in exosome biogenesis.92,94
GM130	Golgi Matrix protein 130 which is a non-exosome protein used as a negative control.95
FLOT1	It stands for Flotillin-1, an exosome marker; it plays a critical role in membrane transport and fusion.92,96
ICAM	It stands for InterCellular Adhesion Molecule and has been found on exosome membranes.97
ALIX	It is endosome associated protein present in most of the exosomes. It plays a role in exosome biogenesis by sorting the exosomal protein cargo.92,98
CD81	It is a tetraspanin that is present in the exosome membrane.92

Table 4 |.

Details of antibodies used in the study

S. No.	Antibody/Fluorochrome	Clonality	Vendor	Host	Specificity Against	Fluorochrome Attached	Dilution Used
1	Anti-GFP antibody	polyclonal	Abcam	Rabbit	All variants of Aequorea victoria GFP such as S65T-GFP, RS-GFP, YFP, CFP, RFP and EGFP	Primary antibody	1:200
2	Goat anti-rabbit IgG	polyclonal	Thermo Fisher Scientific	Goat	Anti-rabbit	Secondary antibody, Alexa Fluor™ 568	1:200
3	Goat anti-mouse IgG	polyclonal	Thermo Fisher Scientific	Goat	Anti-mouse	Secondary antibody, Alexa Fluor™ Plus 405	1:200
4	Anti-HSP90	monoclonal	Abcam	Mouse	Anti-mouse, human, rat, chicken, cow, pig, rabbit	Primary antibody	1:100
5	Anti-RAB7A	monoclonal	Abcam	Rabbit	Anti-mouse, human	Primary antibody	1:200

Key points:

• Exosomes – extracellular vesicles of endosomal origin – are important mediators of cell-cell communications yet are difficult to isolate from the heterogeneous pool of extracellular vesicles using methods based on size, density, and surface markers.

• This protocol employs tissue nanotransfection to deliver reporter constructs driven by cell type-specific promoters for immunomagnetic isolation of specific exosome populations from murine tissue followed by elution with acidic buffer and purification by ultracentrifugation. The resulting exosomes are characterized by established assays.

Data availability

The authors declare that the main data discussed in this protocol are available in the supporting primary research paper (Ref. 53). The snapgene files, QC files, and service reports of the plasmids are available at https://doi.org/10.6084/m9.figshare.26169487.v1 [AU: Please check edits in the ‘Data availability’ statement.]