Protocol to study internalization and localization dynamics of exogenously added proteins in detergent-permeabilized human cells

Summary

Cell-permeabilization and resealing assays are essential tools for studying the localization and functional roles of exogenous factors in detergent-permeabilized cells. We describe a protocol for studying the internalization and localization of exogenous proteins in detergent-permeabilized human cells. It includes detailed steps for culturing, permeabilizing, and preparing ghost cells, followed by lysate incubation and fixation. Subsequent immunofluorescence staining and imaging allow visualization of subcellular protein localization. This assay can be adapted to suit a variety of experimental conditions and research objectives.

For complete details on the use and execution of this protocol, please refer to Ray et al.¹

Graphical abstract

Highlights

• •Instructions to deliver protein or RNA into permeabilized cells using donor cell lysate
• •Steps to culture, permeabilize, and reseal ghost cells with preserved morphology
• •Guidance on lysate incubation and fixation for imaging-based downstream assays
• •Procedures for detecting internalized cargo using immunofluorescence

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Cell-permeabilization and resealing assays are essential tools for studying the localization and functional roles of exogenous factors in detergent-permeabilized cells. We describe a protocol for studying the internalization and localization of exogenous proteins in detergent-permeabilized human cells. It includes detailed steps for culturing, permeabilizing, and preparing ghost cells, followed by lysate incubation and fixation. Subsequent immunofluorescence staining and imaging allow visualization of subcellular protein localization. This assay can be adapted to suit a variety of experimental conditions and research objectives.

Before you begin

The protocol outlined below utilizes HeLa cells as recipient cells and HEK293 cells as donor cells. However, this method has also been successfully validated using additional recipient cell lines, including Huh7 hepatoma cells and differentiated PC12 cells, thereby demonstrating its broader applicability across various cell types.

• 1.
  Prior to seeding recipient cells (post-transfection), coat 18 mm coverslips with a 0.05% gelatin solution.

  • a.
    Autoclave the coverslips in the gelatin solution.
  • b.
    Store them at 4°C.

Note: The coated coverslips can be reused until visible precipitation of gelatin occurs.

• 2.Cultivate both HeLa and HEK293 cells in T25 flasks until they reach 100% confluency. Use the confluent cultures to seed cells into 12-well and 6-well plates, respectively, as required by the experimental protocol.
• 3.Prepare the lysis buffer freshly on the day of the assay. Keep the buffer on ice at all times prior to use to maintain its efficacy and prevent degradation of sensitive components.
• 4.Stock solutions of 2M Sucrose, 100 mM EGTA (pH 7.5), 1 M CaCl₂, 1 M MgCl₂, 2 M KCl, 1 M HEPES (pH 7.5) can be prepared in advance, and stored at 4°C for up to 2–3 months.

Note: Always verify the pH of 1 M HEPES and 100 mM EGTA stock solutions before using them to prepare the lysis buffer.

• 5.Stock solutions of 10 mg/mL Cycloheximide, 0.5 M DTT, and 10 mg/mL Digitonin can also be prepared in advance and stored at −20°C for up to 6–12 months.

Note: Digitonin should be dissolved in dimethyl sulfoxide (DMSO) to ensure complete solubility.

• 6.A 100× stock solution of PMSF (100 mM) must be freshly prepared on the day of the experiment and should not be stored for future use.

Note: PMSF is soluble in DMSO or Isopropanol.

• 7.To ensure experimental consistency and avoid nuclease contamination, use only nuclease-free water, either commercially available ultra-pure water or DEPC-treated water, for the preparation of all buffers. Additionally, always use nuclease-free, commercially available 1× PBS (pH 7.4) whenever PBS is required.

Note: 1X Phosphate-buffered saline (PBS, pH 7.4), used in this study contains sodium chloride (NaCl), potassium chloride (KCl), sodium phosphate dibasic (Na₂HPO₄), and potassium phosphate monobasic (KH₂PO₄).

• 8.Pre-treat glass slides by soaking them in isopropanol to effectively remove any grease or surface contaminants prior to use.

Cell culture and sample preparation

Timing: 3 days

• 9.
  Day 1: Seeding HEK293 cells for transfection.

  • a.
    Culture HEK293 cells in a T25 flask until it reaches approximately 100% confluency (∼2.5 × 10⁶ cells per T25 flask).

    Note: The complete culture medium used to maintain HEK293 cells consists of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. To ensure adequate nutrient supply and optimal cell growth, replace half of the spent medium with fresh complete medium every other day.

  • b.
    Aspirate the spent culture medium from the flask.
  • c.
    Add 1 mL of 1× PBS to wash the cells. Gently swirl or shake the flask for 5 s to remove residual serum, which may inhibit trypsin activity. Discard the PBS wash completely.
  • d.
    Add 500 μL of Trypsin-EDTA to the T25 flask. Gently shake the flask to distribute the solution evenly, and then incubate at 37°C in a CO₂ incubator for 30 s to facilitate cell detachment.
  • e.
    Add 2 mL of fresh complete culture medium to neutralize the trypsin.
  • f.
    Pipette the medium over the cell monolayer to ensure complete detachment and resuspend all cells thoroughly.

    Note: The total volume of the suspension will be 2.5 mL (500 μL Trypsin + 2 mL medium). This results in a final cell concentration of approximately 1 × 10⁶ cells per mL.

  • g.
    Seed 400 μL of the HEK293 cell suspension (approximately 0.4 × 10⁶ cells) into one well of a 6-well plate.
  • h.
    Add 1.6 mL of fresh complete medium to reach a total volume of 2 mL.

    Note: One well of a 6-well plate reaches 100% confluency with approximately 1 × 10⁶ HEK293 cells. This setup ensures ∼80% confluency for optimal transfection efficiency (next day).

    CRITICAL: Ensure that cells used for transfection are within 10 passages post-thaw to maintain consistent growth and transfection efficiency. Always adjust the seeding density based on the actual growth pattern and doubling time of the cells. The above calculations assume a 24-h doubling time.

• 10.
  Day 2: HEK293 cell transfection.

  • a.
    Confirm that HEK293 cells have reached approximately 80% confluency. This confluency ensures optimal conditions for high-efficiency transfection.
  • b.
    Carefully aspirate the existing medium and replace it with antibiotic-free complete medium (DMEM supplemented with 10% FBS, without Penicillin-Streptomycin).

    Note: Antibiotics can interfere with transfection efficiency and cellular uptake of transfection complexes.

  • c.
    Transfect the HEK293 cells with 1 μg of FH-Ago2 plasmid DNA using Lipofectamine 2000 and Opti-MEM as per the manufacturer’s protocol.
    Detailed protocol: Lipofectamine 2000 Reagent Protocol (Thermo Fisher).

    Note: Here, FH-Ago2 has been used as the donor cell derived externally introduced factor. Other HA tagged proteins in the donor cell may be expressed as per the interest of the study.

  • d.
    Allow the cells to incubate with the transfection mixture for 6 h at 37°C in a CO₂ incubator.
  • e.
    After 6 h, carefully aspirate the medium containing the transfection complex and replace it with fresh complete medium supplemented with antibiotics (DMEM + 10% FBS + 1% Penicillin-Streptomycin).

    Note: Do not exceed 6 h of incubation with the transfection reagent, as prolonged exposure can cause cytotoxicity.

    CRITICAL: At near-confluency, HEK293 cells become loosely adherent to the culture surface and are highly susceptible to detachment during medium changes. Always perform medium changes gently and avoid direct pipette contact with the cell monolayer to prevent unintentional cell loss.

• 11.Day 3: HeLa and transfected HEK293 cell seeding for assay.
  For HEK293,

  • a.
    Carefully aspirate the culture medium from transfected HEK293 cells.
  • b.
    Gently rinse the cells with 1× PBS by adding the buffer to each well, swirling for 5–6 s to remove residual serum, and then aspirating it completely.
  • c.
    Add 100 μL of Trypsin-EDTA to each well of 6 well plate. Incubate it at 37°C in a CO₂ incubator for 30 s to facilitate detachment.
  • d.
    Remove the plates from the incubator and immediately add 300 μL of fresh media to HEK293 cells.
  • e.
    Gently pipette up and down to flush the base of the wells and resuspend the cells thoroughly, so that, total 400 μL of cell suspension (Trypsin-EDTA and media) contains 1 × 10⁶ cells.
  • f.
    Transfer the 400 μL HEK293 cell suspension (∼1 × 10⁶ cells) to a 60 mm cell culture dish.
  • g.
    Add 3.6 mL of fresh complete medium to reach a total volume of 4 mL. This seeding ensures that the dish will reach full confluency (∼2 × 10⁶ cells) by the following day, optimal for harvesting.
    For HeLa,
  • h.
    Culture HeLa cells in a separate T25 flask until they reach approximately 100% confluency (∼2.5 × 10⁶ cells per T25 flask).

    Note: The complete culture medium used to maintain HeLa cells consists of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Medium changes were performed every other day, following the same protocol as described for HEK293 cells.

    Note: Handle each cell line independently to prevent any risk of cross-contamination.

  • i.
    Split the cells following the same procedure described for HEK293 cells, adjusting the total volume of the cell suspension to 2.5 mL (500 μL trypsin + 2 mL medium).

    Note: This yields a final cell concentration of approximately 1 × 10⁶ cells/mL.

  • j.
    Take one 0.05% gelatin-coated 18 mm coverslip and place it in a well of a 12-well plate.
  • k.
    Add 150 μL of the HeLa cell suspension (∼0.15 × 10⁶ cells) onto the coverslip.
  • l.
    Add 850 μL of fresh complete medium to bring the final volume to 1 mL.

    Note: One well of a 12-well plate reaches 100% confluency with approximately 0.5 × 10⁶ HeLa cells. This setup allows the HeLa cells on the coverslip to reach approximately 60% confluency (∼0.3 × 10⁶ cells total) by the next day for assay use.

    Note: Make sure to wash each gelatin-coated coverslip with 1× PBS 2–3 times (5 s each wash) to remove excess gelatin before seeding. Keep the coverslips constantly submerged in either PBS or medium to prevent drying. Never allow coverslips to dry, as this may compromise cell attachment and morphology.

    Note: The density of cells in the HeLa culture used for experimentation is important, as a 100% high-density culture of HeLa is associated with a loss of P-bodies. Thus, all experiments must be conducted in cells grown to 60-80% confluency to balance the integrity and number of P-bodies.² In contrast, enhanced cell death occurs when transfection or detergent permeabilization is performed with cells at <40% confluency.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
HA (used in 1:100 dilution)	Roche	11867423001
DDX6 (used in 1:1,000 dilution)	Bethyl	A300-461A
Alexa Fluor 488 (anti-rat) (used in 1:500 dilution)	Invitrogen	A11006
Alexa Fluor 568 (anti-rabbit) (used in 1:500 dilution)	Invitrogen	A11036
Chemicals, peptides, and recombinant proteins
Dulbecco’s modified Eagle’s medium	Gibco	12800-017
Heat-inactivated fetal bovine serum	Gibco	10082-147
Lipofectamine 2000 reagent	Invitrogen	11668-019
Trypsin-EDTA	Gibco	25200-072
1X PBS (pH = 7.4)	Gibco	10010-023
Digitonin	Calbiochem	300410
Sucrose	Sigma	S0389
Ultrapure nuclease-free water	Invitrogen	10977-015
EGTA	Sigma	E8145
Calcium chloride	Merck	C4901
Magnesium chloride	Merck	M8266
Potassium chloride	Merck	P9541
HEPES	Merck	H3537
DTT	Roche	10197777001
PMSF	Omnipure	7110
RNase inhibitor	Thermo Fisher Scientific	N8080119
Paraformaldehyde	Sigma	P6148-500G
Bovine serum albumin	HiMedia	mB083
Goat serum	Invitrogen	16210-064
Triton-X-100	Calbiochem	648468
VECTASHIELD mounting medium with DAPI	Vector Laboratories	(H-1500)
Sodium bicarbonate	Merck	S5761
Gelatin	Merck	9000-70-8
Experimental models: Cell lines
HeLa	ATCC	CCL2
HEK293	ATCC	CRL-1573
Recombinant DNA
F-HA-Ago2	Kind gift from Tom Tuschl	N/A
Software and algorithms
Imaris7	Bitplane	N/A
Other
Microprocessor controlled air system class II biohazard safety cabinet	ESCO	N/A
Light microscope model TS2	Nikon	N/A
CO2 incubator	Innova	N/A
Refrigerated micro centrifuge with 1.5/2 mL tube rotor	Eppendorf	N/A
Platform shaker	Tarsons	N/A
Muta-rotator	N/A	N/A
37°C incubator	Innova	N/A
Vortex	N/A	N/A
Sonicator with timer	N/A	N/A
LSM-40 confocal microscope	Zeiss	N/A
T25 cell culture flasks	Eppendorf	N/A
12 and 6 wells cell culture plate	Tarsons	N/A
60 mm cell culture dish	Nunc	N/A
35 mm Petri dish	Nunc	N/A
Cell scraper	Tarsons	N/A
Microcentrifuge tube	Tarsons	N/A
Micropipette (200 μL–1 mL, 20–200 μL, and 2–20 μL)	Corning	N/A
Micro tips (1 mL, 200 μL, and 20 μL)	Tarsons	N/A
Coverslip and slides	Blue Star	N/A

Materials and equipment

Lysis solution

Chemical	Stock concentration	Final concentration	Volume (in μL)
Sucrose	2 M	0.25 M	125 μL
EGTA (pH 7.5)	100 mM	10 mM	100 μL
CaCl2	1 M	8.4 mM	8.4 μL
MgCl2	1 M	4 mM	4 μL
KCl	2 M	78 mM	39 μL
HEPES (pH 7.5)	1 M	50 mM	50 μL
PMSF	100 mM (100 X)	1 mM (1 X)	10 μL
Cycloheximide	10 mg/mL	100 μg/mL	10 μL
DTT	0.5 M	1 mM	2 μL
RNase Inhibitor	20 U/μL	10 U	0.5 μL
Ultra-pure water	–	–	1000–348.9=651.1∼650 μL

Note: Prepare the lysis buffer freshly on the day of the assay using pre-chilled reagents, and keep the solution on ice at all times. After adding all components, vortex thoroughly to ensure complete mixing. For each assay set, 70 μL of lysis buffer is required to lyse approximately 2 × 10⁶ HEK293 cells. Discard any unused buffer after the assay and do not store for reuse.

Permeabilization solution

Chemical	Stock concentration	Intermediate concentration	Final concentration	Volume (in μL)
Digitonin	10 mg/mL	10 μg/mL (in UP water)	80 ng/mL	8 μL
1 X PBS (pH 7.4)	–	–	–	1000−8 = 992 μL

Note: Prepare the permeabilization solution freshly just before the permeabilization step and keep it on ice until use to maintain its stability and effectiveness. For each coverslip containing approximately 0.3 × 10⁶ recipient HeLa cells, 500 μL of permeabilization solution is required.

Gelatin coating of coverslips (0.05%)

Gelatin (weight in mg)	Distilled water (volume in mL)	Coverslips
25 mg	50 mL	20–30

Note: Dissolve gelatin in distilled water by microwaving the solution for 30–60 s, or until the gelatin is fully dissolved. Using forceps cleaned with 70% ethanol, carefully immerse 20–30 coverslips into the warm gelatin solution. Autoclave the solution along with the coverslips to ensure sterility, then store it at 4°C until the gelatin begins to precipitate out of the solution.

4% Paraformaldehyde solution or fixation solution (100 mL)

Paraformaldehyde (weight in gm)	1X PBS (pH 7.4) (volume in mL)	Other chemicals required
4 g	100 mL	5 M NaOH, Conc. HCl

Note: Take 70 mL of nuclease-free 1X PBS (pH 7.4) in a 100 mL glass bottle and place the bottle in a beaker half-filled with boiling water to provide gentle indirect heating. Slowly add 4 g of paraformaldehyde to the PBS while stirring continuously. If the water in the beaker cools down during the process, reheat it to maintain a warm environment until the paraformaldehyde dissolves completely. Add 5 M NaOH dropwise to the solution until it turns clear, indicating that the paraformaldehyde has fully dissolved. Adjust the pH to 7.4 using concentrated HCl, then bring the final volume to 100 mL with additional 1X PBS. Filter the solution to remove any undissolved particles, and store it in a dark bottle at 4°C for up to 1–2 months.

Blocking solution

Reagent	Percentage	Volume (in μL)
Bovine serum albumin	20%	200 μL
Goat serum	10%	100 μL
Triton-X-100 (10% solution)	0.1%	10 μL
1X PBS (pH 7.4)	–	1000−200−100−10=690 μL

Note: Prepare the blocking solution freshly just before use, and keep it at room temperature to prevent degradation of the detergent. Do not keep the solution at 4°C. For each coverslip containing approximately 0.3 × 10⁶ recipient HeLa cells, 500 μL of blocking solution is required.

Primary antibody solution

Reagent	Percentage or ratio	Volume (in μL)
Bovine serum albumin	20%	40 μL
Anti-HA antibody (Rat)	1:100	2 μL
Anti-DDX6 antibody (Rabbit)	1:1000	0.2 μL
1X PBS (pH 7.4)	80%	200−40=160 μL

Note: Prepare the primary antibody solution freshly just before use and keep it at 4°C until application to preserve antibody activity. For each coverslip containing approximately 0.3 × 10⁶ recipient HeLa cells, 50 μL of primary antibody solution is required.

Secondary antibody solution

Reagent	Percentage or ratio	Volume (in μL)
Bovine serum albumin	20%	40 μL
Anti-Rat antibody with Alexa fluor 488 nm	1:500	0.4 μL
Anti-Rabbit antibody with Alexa fluor 568 nm	1:500	0.4 μL
1X PBS (pH 7.4)	80%	200−40=160 μL

Note: Prepare the secondary antibody solution freshly just before use and keep it at 4°C in the dark to protect the fluorophore from light-induced degradation. For each coverslip containing approximately 0.3 × 10⁶ recipient HeLa cells, 50 μL of secondary antibody solution is required.

Step-by-step method details

Cell-permeabilization and resealing assay

Timing: Approximately2–2.5 h

Timing: Approximately 1 h (for step 1)

Timing: Approximately 30 min (for step 2)

Timing: 30 min (for step 3)

This step involves the permeabilization of recipient cells, allowing for the controlled entry of protein and RNA factors from the donor cell lysate. Following permeabilization, the recipient cells are incubated with the donor cell lysate, facilitating the transfer of cellular components and the resealing of the recipient cells, and finally fixed to preserve cellular architecture, preparing them for the next stage of the protocol—immunofluorescence analysis (Figure 1).

• 1.
  Donor cell lysate preparation:

  • a.
    Prepare the lysis solution as described in the materials and equipment Setup section, ensuring it is freshly made and kept on ice.
  • b.
    Remove the 60 mm culture dish containing approximately 2 × 10⁶ confluent HEK293 cells from the 37°C CO₂ incubator.

    • i.
      Discard the culture medium and gently wash the cells with 1 mL of cold 1X PBS for 5–6 s by rocking the dish.
    • ii.
      Discard the PBS, add 1 mL of fresh cold 1X PBS, and gently scrape the cells from the dish using a sterile cell scraper to ensure complete detachment.
    • iii.
      Transfer the cell suspension into a microcentrifuge tube and immediately place it on ice.
  • c.
    Centrifuge the suspension at 800 × g for 5 min at 4°C to pellet the cells.

    • i.
      Carefully discard the supernatant and resuspend the cell pellet in 70 μL of freshly prepared lysis solution.
    • ii.
      Mix thoroughly to obtain a homogeneous lysate and incubate with gentle rotation for 30 min at 4°C.
  • d.
    Sonicate the lysate using one pulse per second for a total of 12 s.

    • i.
      Centrifuge the sonicated solution at 1000 × g for 10 min at 4°C and carefully collect the clear supernatant (lysate) in a fresh microcentrifuge tube, avoiding any disturbance of the pellet containing cellular debris.
    • ii.
      Determine the protein concentration of the lysate using the Bradford assay.

Note: For a cell input of 2 × 10⁶ HEK293 cells per assay, the protein yield should be approximately 110 μg.

Optional: Keep 10 μL of lysate to check the expression of HA-tagged protein of interest in the lysate by western blotting.

CRITICAL: Since HEK293 cells are very loosely adherent to the base of culture dishes when fully confluent, exercise caution during the washing step to prevent cell dislodgment. Slowly add PBS along the side wall of the dish, ensuring that it doesn't directly contact the cell layer at the bottom. Gently rock the dish in a circular motion to wash the cells without disturbing their attachment. This method minimizes the risk of detaching cells during the procedure.

Pause point: If necessary, the lysate can be kept on ice for up to 30 min before proceeding to the next step. However, it is strongly recommended to continue with the next step immediately to maintain the integrity and activity of the lysate.

• 2.
  Permeabilization of the recipient HeLa cells:

  • a.
    Prepare the permeabilization solution, as described in the materials and equipment Setup section, freshly just before use and keep it on ice to maintain its stability and effectiveness.
  • b.
    Remove the 12-well plate containing coverslips with approximately 0.3 × 10⁶ HeLa cells from the 37°C CO₂ incubator.

    • i.
      Discard the media, add 500 μL of 1X PBS, and gently rock the plate in a circular motion for 5–6 s to wash the cells.
    • ii.
      Discard the PBS and replace it with 500 μL of the freshly prepared permeabilization solution.
  • c.
    Place the plate on a platform shaker at 4°C for 10 min to allow permeabilization.
  • d.
    After 10 min, remove the permeabilization solution and add 500 μL of 1X PBS.

    • i.
      Gently rock the plate in a circular motion for 5-6 s to remove any excess Digitonin.
    • ii.
      Repeat the wash step with fresh PBS, and after the second wash, replace the PBS with 500 μL of fresh 1X PBS.

Note: Always add PBS and the permeabilization solution slowly along the side wall of the well (do not pour directly onto the cell layer), and gently rock the plate in a circular motion to avoid disturbing the cells.

CRITICAL: Do not leave the coverslips in a dry condition at any point during the procedure. Always ensure that the coverslips remain submerged in 1X PBS to prevent the cells from drying out, which could damage or compromise their integrity.

CRITICAL: Do not exceed the 10-min duration for Digitonin permeabilization, as prolonged exposure can negatively impact cell morphology. Since the assay is conducted under live cell conditions, it is crucial to minimize the time between each step. To maintain the integrity of the cells and the accuracy of the results, proceed to the incubation step immediately after completing the permeabilization process, ensuring minimal delay.

• 3.
  Incubation of permeabilized HeLa cells with donor cell lysate:

  • a.
    Prepare a moisture chamber before starting this step.

    • i.
      To create the chamber, take a glass trough and line the base with several layers of tissue.
    • ii.
      Add enough water to the tissue so that it is thoroughly wetted.
    • iii.
      Then, cover the trough with plastic wrap to trap moisture inside.

      Note: This setup ensures that the environment remains humid, preventing the coverslips from drying out during the incubation period.

  • b.
    Clean the tips of a watchmaker’s forceps thoroughly with 70% alcohol and RNase-away solution to ensure no contaminants are present that could interfere with the experiment.
  • c.
    Place a glass plate inside the moisture chamber to provide a stable surface for further steps.
  • d.
    Take a 35 mm Petri dish and place it on top of the glass plate in the moisture chamber.
  • e.
    Add a 70 μL drop of lysate solution to the dish.
  • f.
    Using the prepared forceps, carefully remove the coverslip from the 12-well plate, and place it upside down onto the drop of lysate, ensuring that you avoid trapping any air bubbles beneath the coverslip.
  • g.
    Seal the moisture chamber with plastic wrap to maintain a controlled, moist environment.
  • h.
    Place the chamber in a 37°C incubator and incubate for 30 min (Figure 2).

    CRITICAL: Ensure that the incubation time does not exceed 30 min, as prolonged exposure to the lysate may negatively affect the cell morphology. Since the assay involves live cells, excessive incubation can lead to cell damage. Once the 30-min incubation period is complete, proceed immediately to the fixation step.

    Note: For live cell experiments such as FRAP (Fluorescence Recovery After Photobleaching) or FCS (Fluorescence Correlation Spectroscopy), skip the fixation step and proceed immediately after the incubation period.

Figure 1.

A schematic diagram of the cell-permeabilization and resealing assay done with human cells

HeLa cells were used as recipient cells, and HEK293 cells expressing FH-Ago2 were used for lysate preparation.

Figure 2.

A schematic diagram of the incubation of permeabilized HeLa cells with donor cell lysate

Fixation and immunofluorescence

Timing:1–2 days

Timing: Approximately 50 min (for step 4)

Timing:1–2 days (for step 5)

This step involves the fixation of recipient HeLa cells, followed by immunofluorescence staining to visualize both the internalized donor-derived proteins and endogenous proteins using confocal microscopy. Additionally, this assay can be extended to study RNA dynamics within the recipient cells by employing RNA-FISH (RNA Fluorescence In Situ Hybridization) technique,³ which enables the visualization of internalized RNA molecules at the single-cell level, providing complementary insights alongside protein localization studies.

• 4.
  Fixation:

  • a.
    Remove the moisture chamber from the 37°C incubator and carefully retrieve the coverslip using a clean forceps.
  • b.
    Place the coverslip, cell-side up, into a well of a 12-well plate.
  • c.
    Gently add 500 μL of 1X PBS along the side wall of the well and rock the plate in a circular motion for 5–6 s to remove any residual lysate.

    • i.
      Discard the PBS and repeat the wash once with fresh 1X PBS.
  • d.
    After the second wash, replace the PBS with 300 μL of 4% paraformaldehyde in 1X PBS (fixation solution).

    • i.
      Incubate at room temperature for 30 min on a rocker in dark condition.
  • e.
    After fixation, discard the paraformaldehyde solution and wash the coverslip with 500 μL of 1X PBS for 5 min on a rocker to remove excess fixative.

    • i.
      Repeat this wash step two more times.
    • ii.
      After the third wash, replace the PBS with 500 μL of fresh 1X PBS.

Pause point: At this stage, coverslips can be stored at 4°C for up to 4–5 days. To store, ensure the coverslips are fully submerged in 1 mL of 1X PBS to prevent drying.

• 5.
  Immunofluorescence:

  • a.
    Carefully remove the 1X PBS from the wells.

    • i.
      Add 500 μL of freshly prepared blocking solution onto each coverslip.
    • ii.
      Incubate the 12-well plate on a rocker at room temperature.
  • b.
    Discard the blocking solution and add 500 μL of 1X PBS to each well.

    • i.
      Wash for 5 min on a rocker to remove residual blocking solution.
    • ii.
      Repeat the wash twice more with fresh 1X PBS.
    • iii.
      After the third wash, leave the coverslips in 500 μL of fresh PBS.

      Note: Always add PBS slowly along the well’s side wall to avoid disturbing the cells.

  • c.
    Prepare the previously used moist chamber by placing a 35 mm Petri dish on the glass plate and adding 50 μL of freshly prepared primary antibody solution as a drop in the center.
  • d.
    Using clean forceps, gently lift a coverslip from the 12-well plate and invert it onto the drop of primary antibody solution, ensuring no bubbles form.
  • e.
    Seal the moist chamber with plastic wrap and incubate 16 to 18 h at 4°C.
  • f.
    The next day, carefully flip the coverslip right side up into a clean Petri dish, add 1 mL of 1X PBS, and wash for 5 min on a rocker.

    • i.
      Repeat this wash step twice more, then replace the PBS with 1 mL of fresh PBS.
  • g.
    Prepare a new 35 mm Petri dish in the moist chamber and place 50 μL of freshly prepared secondary antibody solution as a drop in the center.
  • h.
    Gently transfer the coverslip from the previous dish and place it upside down onto the drop of secondary antibody solution.

    Note: Avoid forming air bubbles.

  • i.
    Seal the moist chamber and incubate it at room temperature for 1 h in the dark.
  • j.
    After incubation, flip the coverslip right side up in the Petri dish and add 1 mL of 1X PBS.

    • i.
      Wash for 5 min on a rocker.
    • ii.
      Repeat twice more, and after the final wash, replace with 1 mL of fresh PBS.
  • k.
    Take a clean glass slide that has been stored in isopropanol and dried thoroughly with tissue paper.
  • l.
    Add 5 μL of mounting medium at the center of the slide for each coverslip.
  • m.
    Carefully retrieve the coverslip with forceps and gently place it cell-side down onto the drop of mounting medium on the slide.

    Note: Avoid forming air bubbles.

    Note: From the secondary antibody step onward, perform all steps in a dark environment to protect fluorescent tags.

    Note: Store finished slides at 4°C for short-term use. For long-term storage, keep them at −20°C for up to 3–4 months.

Expected outcomes

A successful Cell-permeabilization-resealing assay serves as a powerful in vitro tool to investigate the molecular interactions between donor-derived factors and endogenous or exogenously expressed components within recipient cells under near-physiological conditions. This technique enables the controlled delivery of specific proteins or RNAs from donor cell lysate into recipient cells that are transiently permeabilized using Digitonin and then resealed to restore membrane integrity. In this setup, the internalized molecules can interact with the recipient cell’s existing components, offering a unique opportunity to study their functional dynamics. In the current study, we describe the co-localization between HA-tagged Ago2 protein, internalized from donor HEK293 cells, and endogenous Rck/p54 protein, which is immunostained in the recipient HeLa cells. Confocal microscopy is used to visualize this interaction, with Rck/p54 serving as a marker for P-bodies, allowing us to assess the targeting and accumulation of Ago2 within cytoplasmic RNA granules. During protocol optimization, we tested various concentrations of digitonin for permeabilizing the recipient cells, as well as different incubation durations with the donor cell lysate (Figures 3A and 3B). We found that treating recipient cells with 80 ng/mL digitonin, followed by a 30-min incubation with the donor cell lysate, allowed for the efficient internalization of donor-derived factors. This condition also supported proper membrane resealing without compromising the morphology or integrity of the recipient cells (Figure 4).

Figure 3.

Effect of the cell-permeabilization and resealing assay on the morphology of recipient HeLa cells

(A) Confocal images showing internalized donor HEK293 cell derived FH-Ago2 protein (green) and endogenous Rck/p54 protein (magenta) in recipient HeLa cells, permeabilized with increasing concentrations of Digitonin (0, 40, 80 and 120 ng/mL) at 4°C for 10 min, incubated with FH-Ago2 expressing donor HEK293 cell lysate at 37°C for 30 min, and immunostained for endogenous Rck/p54.

(B) Confocal images showing Ds Red Actin (magenta) and GFP-SMN protein (green), exogenously expressed in recipient HeLa cells, permeabilized with 80 ng/mL Digitonin at 4°C for 10 min, and incubated with donor HEK293 cell lysate at 37°C for different time points (0, 15, 30, 45 min, 1 h).

Merged images are shown. Scale bar represents 10 μm.

Figure 4.

Effect of cell permeabilization and resealing on the morphology of subcellular structures

Confocal images showing DsRed ER (magenta) and GFP-mito (green) (upper panel) or YFP-endo (green) (lower panel), exogenously expressed in recipient HeLa cells, permeabilized with 80 ng/mL Digitonin at 4°C for 10 min, and incubated with donor HEK293 cell lysate at 37°C for 30 min. Merged images are shown. Scale bar represents 10 μm.

Importantly, this assay system provides substantial flexibility for experimental manipulation. Researchers can modulate the physicochemical environment, such as salt concentration, pH, temperature, or the addition of inhibitors, to examine how these variables influence the internalization process or the extent of protein-protein interactions. It also enables the application of various biological treatments to investigate regulatory mechanisms that govern the localization, post-translational modifications, or functional activity of internalized factors. Using this assay system, we observed that ATP facilitates the compartmentalization of internalized donor cell–derived FH-Ago2 proteins into pre-existing Rck/p54-positive processing bodies (PBs) in recipient HeLa cells (Figure 5). These features make the Cell-permeabilization-resealing assay a valuable method for dissecting complex molecular pathways in a controlled yet biologically relevant setting, as previously demonstrated in Ray et al., 2025.

Figure 5.

Effect of ATP in the compartmentalization of internalized FH-Ago2 protein to Rck/p54-positive P-bodies

Confocal images showing co-localization between internalized donor HEK293 cell derived FH-Ago2 protein (green) and endogenous Rck/p54 protein (magenta) in recipient HeLa cells, permeabilized with 80 ng/mL Digitonin at 4°C for 10 min, incubated with control, 0.5 U/mL Apyrase treated (at 30°C for 30 min) or 1 mM ATP supplemented FH-Ago2 expressing donor HEK293 cell lysate, and immunostained for endogenous Rck/p54.

Merged images are shown. The scale bar of non-zoomed images represents 10 μm, whereas those of zoomed images represent 2 μm.

Quantification and statistical analysis

• 1.
  Quantification of internalized as well as endogenous proteins in the recipient cells using Imaris7 software:

  • a.
    Open Surpass plugin of the Imaris7 software.
  • b.
    Create a surface for the source channel of interest using which the fluorescently labeled protein has been captured.
  • c.
    Set the threshold as per required to erase the background noise and select the actual bodies.
  • d.
    Go to the statistics tab and it will show the total number as well as area of the disconnected components.
• 2.
  Calculation of percentage of internalized proteins co-localizing with endogenous proteins:

  • a.
    Quantify the number of internalized and endogenous proteins.
  • b.
    Calculate the percentage of internalized proteins that colocalize with endogenous proteins by dividing the number of colocalized proteins by the total number of internalized proteins, then multiplying the result by 100 (Table 1).

Note: The coefficient of co-localization between the two proteins can also be measured using the Coloc plugin of Imaris 7 software.

Table 1.

Calculation of the percentage of internalized proteins co-localizing with endogenous protein-formed bodies

Control internalized FH-Ago2	Control colocalized bodies	Ratio	Percentage	Apyrase internalized FH-Ago2	Apyrase colocalized bodies	Ratio	Percentage
10	5	0.5	50	10	2	0.2	20
9	4	0.444444444	44.44444444	7	0	0	0
12	6	0.5	50	7	1	0.142857	14.2857143
11	5	0.454545455	45.45454545	6	1	0.166667	16.6666667
18	7	0.388888889	38.88888889	7	1	0.142857	14.2857143
15	7	0.466666667	46.66666667	12	1	0.083333	8.33333333
14	6	0.428571429	42.85714286	14	0	0	0
9	3	0.333333333	33.33333333	13	0	0	0
6	2	0.333333333	33.33333333	9	0	0	0
6	3	0.5	50	6	0	0	0
5	1	0.2	20	15	1	0.066667	6.66666667
5	2	0.4	40	17	3	0.176471	17.6470588
7	2	0.285714286	28.57142857	13	0	0	0
12	6	0.5	50	11	1	0.090909	9.09090909
14	8	0.571428571	57.14285714	12	0	0	0
14	6	0.428571429	42.85714286	8	2	0.25	25
8	5	0.625	62.5	9	0	0	0
9	4	0.444444444	44.44444444	12	0	0	0
12	8	0.666666667	66.66666667	14	4	0.285714	28.5714286
9	4	0.444444444	44.44444444	16	0	0	0
11	7	0.636363636	63.63636364	11	0	0	0

Limitations

Investigating phase separation in vivo is inherently challenging due to the complexity of the cellular environment, whereas in vitro systems offer the advantage of defined and easily manipulable components. Despite progress, the mechanistic understanding of mRNA compartmentalization, particularly in processing bodies (P-bodies), remains limited. Although recent efforts have successfully reconstituted yeast RNA P-bodies in vitro under near-physiological conditions, the precise role of RNA in P-body assembly is still poorly understood.⁴ To address this, our study aims to develop a controllable in vitro system that closely mimics cellular conditions, enabling detailed investigation of P-body formation, and the influence of external factors. We employed a cell-permeabilization–resealing assay, a powerful method for studying intracellular localization, in which lysates from donor cells, containing diverse proteins and RNAs, are introduced into permeabilized recipient cells, allowing the tracking of internalized factors within a preserved cellular context.^5,6 This assay has previously been adapted to investigate various processes, including the nuclear shuttling of molecular cargoes.^7,8,9,10 In our setup, HEK293 cells served as donors and HeLa cells as recipients, enabling us to examine the localization and behavior of internalized factors while maintaining the natural morphology of the recipient cells and the integrity of existing P-bodies.

Demonstrating phase separation and the subsequent localization of internalized proteins in living cells is essential for understanding the molecular mechanisms that govern protein dynamics. A widely used and potentially effective approach involves live-cell imaging of cells engineered to express an inducible, fluorescently tagged protein (such as GFP-tagged constructs). This method enables real-time visualization of protein internalization and subcellular localization, offering valuable insights into the formation and behavior of these structures under physiological conditions.

However, certain limitations hinder the accuracy of this approach. For instance, leaky expression of GFP in the absence of induction can result in background fluorescence, complicating the distinction between true signal and noise. Moreover, the relatively long half-life of GFP may mask the effects of short-term or transient stimuli, thereby reducing the sensitivity of the assay in assessing rapid changes in protein localization. These challenges can complicate data interpretation and underscore the need for alternative strategies that allow more precise monitoring of protein expression and localization. As such, these factors represent limitations of the assay.

Troubleshooting

Problem 1

Taking out coverslip from a 12-well plate with forceps is critical as it may break. Coverslips are fragile and can easily fracture during handling, especially when being removed from well plates using forceps. Breakage can lead to loss of valuable samples, and hence the need to repeat the assay (during Incubation of permeabilized HeLa cells with donor cell lysate of the Cell-Permeabilization and resealing assay, step f, and Immunofluorescence of the Fixation and Immunofluorescence, step d).

Potential solution

To mitigate the risk of breakage, it is recommended to prepare duplicate coverslips for each assay (during Cell culture and sample preparation, day 3, step j). Having a spare ensures continuity of the experiment even if one coverslip breaks during handling. This proactive step helps avoid delays and the need to restart sample preparation, thus improving experimental efficiency and reliability.

Problem 2

HEK293 cells are loosely adherent to the culture dish surface, making them particularly susceptible to detachment during washing steps (during Donor cell lysate preparation of the Cell-Permeabilization and resealing assay, step b). This unintentional detachment can result in sample loss, variability in cell numbers, and potentially compromised experimental outcomes.

Potential solution

If cells are unintentionally dislodged during the washing step, use a cell scraper to deliberately collect all cells from the dish. Transfer the entire cell suspension to a microcentrifuge tube and centrifuge at 800 × g for 5 min at 4°C to pellet the cells. Discard the supernatant, resuspend the pellet in 500 μL of fresh 1X PBS, and repeat the centrifugation under the same conditions. Proceed to the lysis step with the washed cell pellet.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Suvendra N. Bhattacharyya (sbhattacharyya@unmc.edu).

Technical contact

Technical questions regarding the execution of this protocol should be directed to and will be answered by the technical contact, Dr. Suvendra N. Bhattacharyya (sbhattacharyya@unmc.edu).

Materials availability

This study did not produce any unique reagents.

Data and code availability

• •All data supporting the findings of this study are available from the corresponding authors upon request.
• •This paper does not report the original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

We acknowledge the support of the Council for Scientific and Industrial Research for S.R.’s fellowship. Additionally, S.N.B. was supported by the Swarnajayanti Fellowship (DST/SJF/LSA-03/2014-15) and a High-Risk High-Reward Grant (HRR/2016/000093) from the Department of Science and Technology (DST), Government of India, as well as the CEFIPRA project grant 6003-J. S.N.B. also acknowledges the University of Nebraska, USA’s Start-Up Support Grant. K.M. is supported by the Lieberman Research Award, Department of Anesthesiology, UNMC. We thank the Core Imaging Facility of the CSIR-Indian Institute of Chemical Biology for image capture and analysis.

Author contributions

S.N.B. and K.M. conceived the idea, designed experiments, and analyzed data. S.R. performed the experiments and standardized the assay. S.R. and K.M. wrote the manuscript with S.N.B.

Declaration of interests

The authors declare no competing interests.