Protocol for studying in vitro mouse primary thymocyte differentiation via retroviral-mediated gene expression and stromal-free stimulation

Summary

T cell differentiation and selection in the thymus are pivotal for establishing antigen specificity and shaping the functional repertoire of peripheral T cells. Here, we present an in vitro protocol to investigate the roles of specific target genes during early thymic development in mice utilizing retroviral vectors. We describe steps for retroviral packaging, the isolation of primary murine thymic cells, retroviral transduction, and the subsequent in vitro differentiation of thymocytes.

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

Graphical abstract

Highlights

• •Instructions for the isolation of mouse DN3 thymocytes
• •Guidelines for retroviral-mediated gene expression in thymocytes
• •Procedure for stromal-free in vitro differentiation of mouse DN3 thymocytes

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

T cell differentiation and selection in the thymus are pivotal for establishing antigen specificity and shaping the functional repertoire of peripheral T cells. Here, we present an in vitro protocol to investigate the roles of specific target genes during early thymic development in mice utilizing retroviral vectors. We describe steps for retroviral packaging, the isolation of primary murine thymic cells, retroviral transduction, and the subsequent in vitro differentiation of thymocytes.

Before you begin

T cell development is a tightly regulated and intricate process. Hematopoietic stem cells, originating from the bone marrow, travel to the thymus where they differentiate into precursor cells capable of becoming T cells.² During this phase, these precursor cells lack the surface markers CD4 and CD8, thus being classified as double-negative (DN) cells. Although DN cells represent a small proportion of total thymocytes, this phase plays a crucial role in the commitment to the T cell lineage and in the β-selection process.

DN thymocytes are subdivided into four distinct developmental stages based on the expression patterns of the CD44 and CD25 markers: DN1 (CD44⁺CD25^-), DN2 (CD44⁺CD25⁺), DN3 (CD44-CD25⁺), and DN4 (CD44^-CD25^-). The Notch signaling pathway is pivotal in regulating T cell lineage differentiation at each of these stages.³ In addition to cell-intrinsic cues, chemokines play a pivotal role in thymocyte development by orchestrating their spatially regulated migration within the thymic microenvironment. Among these, CXCL12 is the first chemokine identified as essential for the proliferation and differentiation of DN3 thymocytes.^4,5 This function is mechanistically dependent on the coordinated integration of signaling from the Notch pathway and the pre-TCR, underscoring a tightly regulated signaling network during early T-cell development.⁶ Elucidating these molecular mechanisms has facilitated the establishment of a defined in vitro culture system that combines Notch activation—via OP9-DL1 stromal cells or DLL4-coated surfaces—with CXCL12 stimulation to support early T-cell differentiation, thereby providing a robust experimental platform for investigating this developmental process.

Our protocol provides a comprehensive demonstration of employing retroviruses to induce gene overexpression in primary thymocytes in vitro. This technique enables rapid and efficient investigation of the effects of target genes on early thymic development. This protocol can also be adapted for studying the role of specific genes in the differentiation of peripheral mature T cells. However, modifications to both the retroviral transduction conditions and the subsequent T cell differentiation culture conditions are required.⁷ Furthermore, our in vitro differentiation protocol of thymic DN3 cells employs a DLL4-coated plate stimulation. In contrast to the traditional OP9-DL1 cell co-culture stimulation approach, this method does not introduce additional cell types, thereby resulting in a cleaner differentiation system and more suitable for retroviral infection. Moreover, the differentiation protocol outlined here for DN3 thymocytes is also directly applicable to analysis of differentiation in DN thymocyte populations.

Innovation

The innovation of this study lies in the strategic integration and optimization of retroviral transduction with DLL4-mediated Notch activation into a single, stromal-free in vitro system for thymocyte differentiation. This streamlined approach eliminates the dependence on complex stromal cocultures, thereby reducing biological variability and improving experimental reproducibility. By enabling direct and precise assessment of gene function within a unified workflow, the protocol provides a simplified yet robust platform suitable for high-throughput genetic and functional screening. Overall, this methodological advancement offers a standardized and scalable system that facilitates studies on T-cell development and related immunological processes.

Institutional permissions (if applicable)

All mice were bred and maintained under specific-pathogen-free (SPF) conditions at the Zhejiang University (ZJU) Animal Center. All experimental procedures were performed in accordance with the guidelines established by the ZJU Institutional Animal Care and Use Committee (IACUC) and were approved under protocol number ZJU20230095.

Mice

Timing: 4–6 weeks

In this protocol, 4 to 6 weeks old mice are employed for the isolation of DN thymocytes, as using animals outside this age range leads to decreased isolation efficiency. Troubleshooting 3.

Core plasmid construction

Timing: 4.5–5 days

Timing: 10 min (for step 1)

Timing: 10 min (for step 2)

Timing: 5 h (for step 3)

Timing: 2 h (for step 4)

Timing: 30 min (for step 5)

Timing: 3.5 days (for step 6)

This protocol utilizes the retroviral vector pMX-IRES-RFP as the backbone vector. The overall construction process of the plasmid is outlined in Figure 1A.

• 1.Select clone sites to linearize the vector. Keep the GC content of the 20-bp regions flanking the cloning site between 40% and 60% to maximize cloning efficiency.

Note: It is recommended to select regions with minimal repetitive sequences and a uniform GC content for cloning.

• 2.
  Use the CE Design online tool for primer design to amplify the insert fragment with homologous sequences.

  • a.
    Select single-fragment insertion strategy as the clone strategy.
  • b.
    Linearized pMX-IRES-RFP by PCR.
  • c.
    Input the following DNA sequences into the tool to generate the primer sequences: the upstream vector sequence (5′ to 3′, sense strand, ≥20 bp) and the downstream vector sequence (5′ to 3′, sense strand, ≥20 bp) flanking the cloning site, along with the full insert sequence (5′ to 3′, ≥50 bp).

    • i.
      Use the mouse IRF4 gene as an example (Figure 1B). The forward primer for the insert is: IRF4-F: 5′-CTAGACTGCCGGATCTAGCTTGAACTTGGAGACGGGCAGC-3′; the reverse primer for the insert is: IRF4-R: 5′-ACTGGGATCCTTAATTAACTTCACTCTTGGATGGAAGAATGACG-3′. The forward primer for the cloning vector is: common-F: 5′-AGTTAATTAAGGATCCCAGTGTGGTG-3′, and the reverse primer for the cloning vector is: common-R: 5′-AGCTAGATCCGGCAGTCTAGAGG-3′.
• 3.Obtain linearization vector by inverse PCR amplification of the circular plasmid template using a high-fidelity DNA polymerase (see Table 1 for the reaction components and cycling conditions).
• 4.Amplify the DNA insert fragment following the conditions outlined in Table 2 similarly.
• 5.Analyze and purify the PCR products using gel electrophoresis, yielding a 5,950-bp linearized plasmid and a 1,390-bp insert fragment containing the homologous arms.

Note: It is crucial to control the amount of circular plasmid template to 0.1–1 ng in the PCR system to minimize residual template DNA, which could negatively affect cloning efficiency.

• 6.
  Purify the PCR product using a gel extraction kit, then circularize the plasmid using the CloneExpress MultiS One Step Cloning Kit. The recombination products can then be used for transformation assays:

  • a.
    Transform the homologous recombination product into E. coli DH5α cells.

    Note: For viral vectors containing long terminal repeats (LTRs), the Escherichia coli strain Stbl3 is preferred, as it minimizes sequence instability and prevents the loss or rearrangement of repetitive elements.

  • b.
    Select single colonies and culture them for 16–18 hours in shaking culture at 37°C.
  • c.
    Extract plasmid DNA from the culture and submit it for sequencing. After confirming the correct sequence, amplify the plasmid for further experiments.

    CRITICAL: High-quality plasmid DNA (≥1 μg/μL) is essential for efficient downstream viral packaging. Troubleshooting 2.

Figure 1.

Plasmid Construction and Transfection Efficiency in Plat-E Cells

(A) Schematic of plasmid construction via homologous recombination. The template plasmid was linearized by PCR using primers containing homologous arm sequences. The insert fragment, flanked by identical homology arms, was amplified in a separate PCR reaction. The linearized vector and insert fragment were subsequently assembled into a circular recombinant plasmid using a commercial homologous recombination kit.

(B) Map of the successfully constructed plasmid. The pMX-IRES-RFP vector serves as the initial backbone, with IRF4 used as an example of the inserted sequence. The locations of the primer binding sites are indicated by circles. The common-F/R primers are being used for plasmid linearization, whereas the IRF4-F/R primers are designed to amplify the insert fragment. The full names of the abbreviations shown on the plasmid are as follows: AmpR, ampicillin resistance gene; LTR, long terminal repeat; MMLVψ, packaging signal of Moloney murine leukemia virus; gag, truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon.

(C) Transfection efficiency in Plat-E cells. Representative images of Plat-E cells 72 hours after transfection with the pMX-IRES-RFP plasmid. The bright-field image (left) displays cellular morphology and confluency, while the red fluorescent protein (RFP) signal (right) indicates successful transfection. Scale bar: 10 mm.

Table1.

PCR Reaction Components and PCR Program for linearize vector construction

PCR reaction components		PCR program
Component	Volume (μL)	Step	Temp.	Time	Cycle
2 × Phanta Max buffer	25	1	95°C	3 min	1
dNTP mix(10 mM each)	1	2	95°C	15 s
IRF4-F (10 μM)	2	3	65°C	15 s
IRF4-R (10 μM)	2	4	72°C	6 min	Go to Step 2 35×
Phanta Max Super-Fidelity DNA polymerase	1	5	72°C	5 min	1
Template (0.1-1ng)	X	6	10°C	∞
ddH20	up to 50

Table2.

PCR Reaction Components and PCR Program for Insert sequence construction

PCR reaction components		PCR program
Component	Volume (μL)	Step	Temp.	Time	Cycle
2 × Phanta Max buffer	25	1	95°C	3 min	1
dNTP mix(10 mM each)	1	2	95°C	15 s
common-F (10 μM)	2	3	65°C	15 s
common-R (10 μM)	2	4	72°C	2 min	Go to Step 2 35×
Phanta Max Super-Fidelity DNA polymerase	1	5	72°C	5 min	1
Template (50ng)	X	6	10°C	∞
ddH20	up to 50

Cell culture

Timing: 30 min

Timing: 5 min (for step 7)

Timing: 5 min (for step 8)

Timing: 2 min (for step 9)

Timing: 8 min (for step 10)

Timing: 5 min (for step 11)

Timing: 4 min (for step 12)

Timing: 1 min (for step 13)

Platinum-E (Plat-E) is a highly efficient retrovirus packaging cell line derived from human HEK-293T cells.⁸ These cells are maintained in Plat-E cell culture medium at 37°C in a humidified (90%–95% humidity) incubator with 5% CO₂. They are routinely passaged every 2–3 days or upon reaching 80 confluences to ensure continuous logarithmic growth and to maintain their retroviral packaging competency. The subculture process was performed as follows.

• 7.Aspirate the used culture medium carefully and wash cells twice with 2 mL of sterile phosphate-buffered saline (PBS) to remove residual medium.
• 8.Add 1 mL of trypsin solution and incubate the culture vessel at 37°C in a 5% CO₂ incubator for 2 minutes. Monitor under microscope until cells detach and become suspended.
• 9.Add 3 mL of pre-warmed (37°C) Plat-E cell culture medium to neutralize trypsin activity. Gently pipette the suspension to ensure even distribution.
• 10.Transfer the entire suspension to a 15 mL sterile centrifuge tube. Centrifuge at 500 × g for 5 minutes at 4°C to pellet cells.
• 11.Carefully aspirate the supernatant without disturbing the cell pellet. Resuspend the pellet in 1 mL of pre-warmed Plat-E cell culture medium by gentle pipetting or vortexing to obtain a single-cell suspension.
• 12.Transfer an appropriate volume of cell suspension to a cell culture dish according to the desired split ratio. Add pre-warmed Plat-E cell culture medium to achieve the final working volume. Gently rock the dish to ensure even cell distribution.
• 13.Place the culture dish in a humidified incubator maintained at 37°C with 5% CO₂ for continued cultivation.

Note: For critical virus production experiments, only cells within a validated passage range (P3 to P10) were used to guarantee high transfection efficiency and consistent viral titers. Troubleshooting 2.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Pacific Blue anti-mouse CD4 (Dilutions: 1:500)	BioLegend	Cat# 116008; RRID: AB_11149680
Brilliant Violet 650 anti-mouse CD8α (Dilutions: 1:500)	BioLegend	Cat# 100742; RRID: AB_2563056
PE anti-mouse CD8β (Dilutions: 1:500)	eBioscience	Cat# 12-0083-83; RRID: AB_657768
FITC anti-mouse/human CD44 (Dilutions: 1:500)	BioLegend	Cat#103005 RRID: AB_312956
APC anti-mouse/human CD44 (Dilutions: 1:500)	BioLegend	Cat# 103012; RRID: AB_312963
PE-Cy7 anti-mouse CD25 (Dilutions: 1:500)	BioLegend	Cat# 102016; RRID: AB_312865
FITC anti-mouse Lineage Cocktail (Dilutions: 1:200)	BioLegend	Cat# 133302; RRID: AB_10697030
Biotin anti-CD4 (Dilutions: 1:100)	BioLegend	Cat# 100403; RRID: AB_312688
Biotin anti-CD8α (Dilutions: 1:100)	BioLegend	Cat# 100704; RRID: AB_312742
Bacterial and virus strains
DH5α	Vazyme	Cat#C502-02
Biological samples
4-6 weeks old mouse thymus tissue (C57BL/6)	ZJU Animal Center	N/A
Chemicals, peptides, and recombinant proteins
CXCL12	Novoprotein	Cat#C698
DLL4-Fc	R&D Systems	Cat#10089-D4
Zombie Aqua Fixable Viability Kit	BioLegend	Cat# 423102
Polybrene	Sigma-Aldrich	Cat# H9268
Puromycin	Beyotime	Cat# ST551-10mg
Blasticidin	Beyotime	Cat# ST018-1mL
ExFect Transfection Reagent	Vazyme	Cat# T101-01
Phanta Max Super-Fidelity DNA polymerase	Vazyme	Cat#P505
Critical commercial assays
MojoSort Streptavidin Nanobeads	BioLegend	Cat# 480016
cloneExpress MultiS One Step Cloning Kit	Vazyme	Cat# C113
TIANprep Midi Plasmid Kit	TianGen	Cat# DP106-02
TIANgel Purification kit	TianGen	Cat# DP219-03
Experimental models: Cell lines
Platinum-E (Plat E) cells	A gift from professor Lie Wang	N/A
NIH 3T3 cells	A gift from professor Lie Wang	N/A
Experimental models: Organisms/strains
4–6 weeks old C57/BL6J wild-type mice, female	Gempharmatech	Cat# N000013
Recombinant DNA
pMX-IRES-RFP	This paper	N/A
Software and algorithms
FlowJo 10.10.0	FlowJo, LLC	https://flowjo.bectondickinson.cn/
Figdraw 2.0	Home for Researchers	https://www.figdraw.com/
Graphpad Prism 10.2.0	GraphPad Software	https://www.graphpad.com/
Other
FACS analyzer LSR Fortessa	BD Biosciences	N/A
FACS sorter BD FACSAria II	BD Biosciences	N/A
FACS analyzer ACEA NovoCyte	Agilent Technologies	N/A
Microscopy CKX53	Olympus	N/A
Centrifuge	Thermo Scientific	Fresco 17
15 mL centrifuge tubes	NEST	601001
50 mL centrifuge tubes	NEST	601002
6 cm cell culture dish	NEST	705201
1.5 mL microcentrifuge tubes	NEST	615601
Cell strainers	Biosharp	BS-40-XBS

Materials and equipment

FACS Buffer

Reagent	Final concentration	Amount
Fetal Bovine Serum	2%	1 mL
EDTA (2 M)	2 mM	50 μL
PBS	N/A	48.5 mL
Total	N/A	50 mL

Store at 4°C for up to 1 month.

Thymic DN Cell Culture Medium

Reagent	Final concentration	Amount
Fetal Bovine Serum	10%	5 mL
penicillin-streptomycin	1%	500 μL
HEPES (1 M)	10 mM	500 μL
sodium pyruvate (10 mM)	1 mM	500 μL
β-mercaptoethanol (50 mM)	0.5 mM	500 μL
α-MEM medium	N/A	43 mL
Total	N/A	50 mL

Store at 4°C for up to 1 month.

Plat-E Cell Culture Medium

Reagent	Final concentration	Amount
Fetal Bovine Serum	10%	5 mL
penicillin-streptomycin	1%	500 μL
puromycin	10 μg/mL	50 μL
blasticidin	10 μg/mL	50 μL
DMEM medium	N/A	44.5 mL
Total	N/A	50 mL

Store at 4°C for up to 1 month.

Red Blood Cell Lysis Buffer

Reagent	Final concentration	Amount
NH4Cl	10%	1.8675 g
Tris	1%	0.65 g
ddH2O	N/A	250 mL
Total	N/A	250 mL

Store at 4°C for up to 3 months.

CRITICAL: Adjust the pH to 7.2. Filter through a 0.22 μm filter.

Step-by-step method details

Packaging and harvesting of retrovirus

Timing: 7.5 days

Timing: 18 h (for step 1)

Timing: 3 days (for step 2)

Timing: 10 min (for step 3)

Timing: 4 days (for step 4)

This step details the experimental procedure and key precautions for retrovirus production and collection.

• 1.
  Plate packaging cell Plat-E cells one day before transfection.

  • a.
    Maintain Plat-E cells in a 6 cm cell culture dish using Plat-E cell culture medium without puromycin and blasticidin. When the confluence become 80%–90%, Trypsinize and centrifuge them at 800 × g for 5 min.
  • b.
    Passage the cells at the ratio of 1:3 to 1:4 into a new 6 cm dish. Gently swirl the dish to ensure even distribution of cells.
  • c.
    Incubate the dish for 16–18 hours at 37°C.
  • d.
    The next day, examine cell density and attachment status. Proceed to transfection when cells are well-attached and reach approximately 60%–70% confluency.

CRITICAL: The Plat-E cell culture medium should be warmed at 37°C to get a better cell viability. To maximize transfection efficiency and maintain optimal cell viability, the packaging cells were transferred to medium without puromycin and blasticidin before transfection. Troubleshooting 1.

• 2.
  Transfect the plasmid.

  • a.
    Prepare two 1.5 ml microcentrifuge tubes to make up transfection complex mix A and mix B.
  • b.
    Add 200 μL of OPTI-MEM medium to each tube. To mix A: add 12 μL of transfection reagent and mix gently. To mix B: Add 6 μg of core plasmid DNA (pMX-IRES-RFP) and mix gently.
  • c.
    Transfer the mix A dropwise into mix B and mix thoroughly by gentle pipetting.
  • d.
    Keep the mixture to incubate at room temperature (23°C–25°C) for 15–20 minutes.
  • e.
    Gently add the mixture dropwise to the Plat-E cells, swirling the plate lightly to achieve uniform distribution. Incubate the cells at 37°C with 5% CO₂.
  • f.
    After 4-6 hours, replace the medium with fresh Plat-E cell culture medium to reduce cytotoxity.
  • g.
    Replace the medium again with fresh Plat-E cell culture medium after 24 hours post-transfection to enhance viral titer.
  • h.
    Observe the percentage of RFP-positive cells under microscopy to determine transfection efficiency (Figure 1C).

CRITICAL: When changing the medium, handle gently to avoid dislodging the cells, as this will significantly reduce retroviral titer. Troubleshooting 2.

• 3.
  Collect the retroviral.

  • a.
    Collect the virus-containing supernatant between 48 and 72 hours post-transfection prior to the acidification-induced color shift of the culture medium to yellow. Troubleshooting 5.
  • b.
    Filter the supernatant through a 0.45 μm filter.
  • c.
    Use the filtered viral supernatant immediately or store it at −80°C for future use.

Note: Avoid repeated freeze-thaw cycles of the viral stock. Troubleshooting 4.

• 4.
  Titrate the retroviral post-collection.

  • a.
    Plate 50000 NIH3T3 cells in 24-well plate using complete DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. Incubate the plate for 16–20 hours at 37°C.
  • b.
    Trypsinize 3 wells from plate to determine the average number of cells per well at the time of infection.

    Note: This value is used to calculate the total number of target cells for viral titer determination.

  • c.
    Prepare a series of 10-fold dilution of the virus supernatant in DMEM. Each dilution is freshly made prior to infection to ensure accuracy.
  • d.
    Replace old DMEM cell culture medium with 450 μL DMEM with 8 μg/mL polybrene.

    • i.
      Add 50 μL/well of the diluted viral suspensions to the corresponding wells in triplicate.
    • ii.
      Control wells receive 50 μL/well DMEM only and serve as mock-infected samples. Gently swirl the plate to ensure mixing.
    • iii.
      Return the plate to the 37°C CO₂ incubator for 24 hours.
  • e.
    replace medium containing the viral supernatant with 500 μL fresh DMEM cell culture medium.
  • f.
    48 hours after infection, trypsinize and resuspend the NIH3T3 cells from each well in FACS buffer individually. Analyze the percentage of RFP⁺ cells (% RFP⁺) using Flow Cytometer.
  • g.
    Calculate the viral titer using the data from the dilution where the percentage of positive cells is between 2% and 20% to ensure a linear relationship. The following formula is applied: Viral Titer (TU/mL) = (% RFP⁺×total cell number× dilution factor)/0.05 mL.

Isolate the mice DN3 thymocytes

Timing: 3–4 h

Timing: 30 min (for step 5)

Timing: 10 min (for step 6)

Timing: 30 min (for step 7)

Timing: 30 min (for step 8)

Timing: 1.5 h (for step 9)

This step describes the procedure for isolation of mouse DN3 thymocytes, including thymus harvesting, single cell suspension preparation, enrichment of DN cells, and sorting of DN3 cells.

• 5.
  Aseptic work surfaces and pre-chilled reagents were prepared prior to dissection.

  • a.
    Pre-chill a 6-well plate by adding 2 mL of FACS buffer per well and placing it on ice.
  • b.
    Disinfect dissection instruments and the biosafety cabinet (BSC) work surface by wiping with 75% ethanol.
  • c.
    Place instruments inside the BSC and expose the work area to UV irradiation for 15 minutes to ensure sterility.
• 6.
  Excise the thymus from the thoracic cavity and rinse to remove blood.

  • a.
    Euthanize the mouse in accordance with approved institutional protocols.
  • b.
    Using sterile scissors, carefully open the anterior rib cage to expose the thoracic cavity. The thymus can then be identified in the upper mediastinum, situated anterior to the heart.
  • c.
    Grasp the thymic base gently with forceps. Carefully dissect the entire thymic lobes free from surrounding connective tissue and mediastinum, avoiding damage to major vessels to minimize bleeding.
  • d.
    Rinse the excised thymus thoroughly in FACS buffer to remove residual blood.
  • e.
    Transfer the thymus to a well of the pre-chilled 6-well plate.
• 7.
  Dissociate the thymus into single cell suspension. Troubleshooting 3.

  • a.
    Place the thymus between two ground-glass slides. Apply gentle pressure and grind the tissue using a circular motion. Avoid excessive force, as it can damage the cells.
  • b.
    Thoroughly rinse the slides with FACS buffer to collect all dissociated cells into the corresponding well.
  • c.
    Clean the glass slides meticulously with ethanol and subsequently rinse with FACS buffer between processing different thymic samples to prevent cross-contamination.
  • d.
    Gently pipette the tissue suspension in the well up and down several times using a pipette to ensure homogeneity and break up large clumps.
  • e.
    Filter the suspension through a sterile 70-μm (300-mesh) cell strainer into a sterile 15-mL tube placed on ice.
  • f.
    Keep the filtered single-cell suspension on ice until further processing.
  • g.
    Erythrocyte Lysis perform ONLY if the initial thymic suspension contains significant erythrocyte contamination (visible red pellet).

    • i.
      Centrifuge the single-cell suspension at 500 × g for 5 minutes at 4°C. Completely remove and discard the supernatant.
    • ii.
      Resuspend the cell pellet in 2 mL of pre-chilled red blood cell lysis buffer. Mix gently by pipetting.
    • iii.
      Incubate at room temperature (23°C–25°C) for 1-2 minutes. Do not exceed 2 minutes. Add 4 mL of ice-cold FACS buffer to stop lysis. Mix immediately.
    • iv.
      Centrifuge at 500 × g for 5 minutes at 4°C. Aspirate and discard the supernatant carefully.
    • v.
      Resuspend the pellet in 1 mL of ice-cold FACS buffer. Keep the suspension on ice until further use.
• 8.
  Enrich DN cells via negative selection using biotinylated antibodies and streptavidin nanobeads to deplete DP and SP thymocytes.

  • a.
    Adjust the concentration of the thymic single-cell suspension to 1×10⁷ cells/mL using FACS buffer.
  • b.
    Transfer 1 mL aliquots of the suspension into individual 5-mL flow cytometry tubes.
  • c.
    Add 5 μg of biotin-conjugated anti-CD4 antibody and 5 μg of biotin-conjugated anti-CD8α antibody per mL of cell suspension. Mix the suspension thoroughly to ensure uniform antibody distribution.
  • d.
    Incubate the tubes on an orbital shaker at 80 rpm and 4°C for 15 minutes.
  • e.
    Add the appropriate volume of MojoSort Streptavidin Nanobeads to each tube according to the manufacturer’s instructions. Mix the suspension thoroughly.
  • f.
    Incubate the tubes on the orbital shaker at 150 rpm and 4°C for 5 minutes.
  • g.
    Add 2 mL of FACS buffer to each tube and mix thoroughly. Place the tubes on a magnetic stand. Allow the bead-bound complexes to separate at room temperature (23°C–25°C) for 3 minutes.
  • h.
    Carefully transfer the supernatant (containing the unbound, enriched DN cells) to a new flow cytometry tube. The resulting DN-enriched cell suspension is now ready for subsequent sorting experiments. See Figure 2B as an example.
• 9.
  Isolate the DN3 population by flow cytometry using a surface marker staining strategy to identify DN3 thymocytes.

  • a.
    Perform surface staining on the enriched DN cell suspension.

    • i.
      Count the enriched DN cells and adjust concentration to 1×10⁷ cells/mL using FACS buffer.
    • ii.
      Add 0.4 μL of Fc block per 100 μL cell suspension. Incubate protected from light at 4°C for 10 minutes to block non-specific Fc receptor binding.
    • iii.
      Add the following antibodies per 100 μL cell suspension: 0.5 μL FITC-conjugated Lineage Cocktail (1:200, anti-CD3ε, anti-Gr-1, anti-B220, anti-TER-119, anti-CD11b), 0.2 μL Pacific Blue (PB)-conjugated anti-CD4 (1:500), 0.2 μL Phycoerythrin (PE)-conjugated anti-CD8β (1:500), 0.2 μL Allophycocyanin (APC) -conjugated anti-CD44 (1:500), 0.2 μL PE-Cy7-conjugated anti-CD25 (1:500).
    • iv.
      Mix thoroughly and incubate protected from light at 4°C for 30 minutes.
    • v.
      Add twice the tube volume of FACS buffer. Centrifuge at 500 ×g for 5 minutes at 4°C. Aspirate the supernatant completely.

      Note: Ensure the use of antibodies against CD8α and CD8β for biotinylated enrichment and flow cytometric sorting staining, respectively.

  • b.
    Resuspend the cell pellet in FACS buffer at 1×10⁷ cells/mL. Filter the suspension through a 70-μm (300-mesh) cell strainer immediately prior to sorting to prevent nozzle clogging.
  • c.
    Set gates to sort the Lin^-CD4^-CD8^-CD44^-CD25⁺ cell population (DN3 stage). Collect sorted cells in tubes containing collection buffer (thymic DN cell culture medium with double FBS). See Figure 2A as an example.
  • d.
    Place collected cells on ice for 60 minutes post-sort. Centrifuge at 500 ×g for 20 minutes at 4°C.
  • e.
    Proceed with downstream applications using the pelleted DN3 cells.

    CRITICAL: Allow the post-sort rest period to dissipate electrostatic charge repulsion between cells, enabling efficient pelleting and maximizing yield. Troubleshooting 3.

Figure 2.

Isolation of DN3 cells

(A) Flow cytometry gating strategy for the isolation of DN3 cells.

(B) The left panel shows enriched thymocytes used to obtain DN cells with a purity of ≥90% and a residual DP cell population of <1%. The right panel displays the subsequent sorting of DN cells to isolate DN3 cells with a final purity of ≥99%.

Retroviral transduction of DN3 cells

Timing: 1.5 days

Timing: 18 h (for step 10)

Timing: 18 h (for step 11)

Timing: 6 h (for step 12)

This step describes the procedure for transduce activated DN3 cells using spin transfection with retroviral supernatant following pre-stimulation.

• 10.
  Coat 48-well plates with DLL4 for 16–18 hours.

  • a.
    Dilute recombinant DLL4 protein to 2 μg/mL in sterile Phosphate-Buffered Saline (PBS). Add 300 μL of the DLL4 solution per well of a 48-well plate.
  • b.
    Seal the plate edges with Parafilm. Incubate the plate at 4°C for 16–18 hours.
• 11.
  Activate sorted DN3 cells for retroviral susceptibility.

  • a.
    Resuspend freshly sorted DN3 cells in DN3 culture medium by gently pipetting the solution 5-8 times using a wide-bore pipette tip to minimize mechanical shear stress. Aspirate PBS completely from the DLL4-coated wells.
  • b.
    Seed 5 × 10⁵ DN3 cells per well in 300 μL DN3 culture medium. Add recombinant CXCL12 to a final concentration of 100 ng/mL per well. Troubleshooting 4.
  • c.
    Incubate the plate at 37°C, 5% CO₂ for approximately 18 hours.

Note: The hallmark of successful DN3 cell activation is a 20% increase in cell diameter compared to the unactivated cells.

• 12.
  Perform retroviral transfection via centrifuge.

  • a.
    Aspirate the activation medium completely from each well. Add 1 mL of freshly prepared retroviral supernatant (previously generated) per well.
  • b.
    Supplement each well with 10 mM HEPES to maintain a stable pH (7.0–7.6) and 10 μg/mL polybrene.
  • c.
    Mix gently by swirling the plate. Seal the entire plate securely with Parafilm.
  • d.
    Centrifuge the sealed plate at 800 ×g for 3 hours at 20°C (set centrifuge acceleration/brake to ramp 3).
  • e.
    Remove the plate carefully from the centrifuge. Incubate the plate undisturbed at 37°C, 5% CO₂ for 4–6 hours.
  • f.
    Proceed with DN3 cell differentiation culture protocols. Troubleshooting 4.

CRITICAL: Avoid positioning cells in perimeter wells of the 48-well plate during centrifugation, as this may compromise cell viability and growth due to edge effects. Troubleshooting 5.

DN3 cell differentiation and flow cytometric analysis

Timing: 2–3 days

Timing: 18 h (for step 13)

Timing: 2.5 days (for step 14)

Timing: 30 min (for step 15)

Timing: 30 min (for step 16)

This step describes the procedure for Assess retroviral transduction efficiency and DN3 differentiation status after 48-hour culture on DLL4-Fc-coated plates by flow cytometry.

• 13.
  Coat 96-well plates with DLL4-Fc for 16-18 hours.

  • a.
    Dilute recombinant DLL4-Fc protein to 2 μg/mL in sterile PBS. Add 100 μL of the DLL4-Fc solution per well of a 96-well plate.
  • b.
    Seal the plate edges with Parafilm. Incubate the plate at 4°C for 16–18 hours.
• 14.
  Seed transduced DN3 cells for differentiation.

  • a.
    Resuspend the post-transduction DN3 cells in DN3 culture medium at a concentration of 1 × 10⁶ cells/mL.
  • b.
    Aspirate the coating solution completely from the DLL4-Fc-coated 96-well plate. Add 200 μL of the DN3 cell suspension per well. Add recombinant CXCL12 to a final concentration of 100 ng/mL per well.
  • c.
    Incubate the plate at 37°C, 5% CO₂ for 48–72 hours. Troubleshooting 5.
• 15.
  Harvest cells and perform surface staining for analysis.

  • a.
    Collect cells from the 96-well plate.
  • b.
    Perform surface antibody staining exactly as described in the previous “isolate the mice DN3 thymocytes” protocol (9.a).
• 16.
  Analyze transduction efficiency and differentiation by flow cytometry (Figure 3A).

  • a.
    Acquire stained cells on a flow cytometer.
  • b.
    Determine transduction efficiency: Calculate the percentage of cells positive for the fluorescent marker (e.g., RFP) encoded by the retroviral core plasmid.
  • c.
    Assess DP differentiation: Calculate the ratio of CD4⁺CD8⁺ (DP) cells to CD4^-CD8^- (DN) cells.
  • d.
    Assess DN4 differentiation: Calculate the ratio of CD44^-CD25⁺ (DN3) cells to CD44^-CD25^- (DN4) cells.

Figure 3.

Representative illustration of effective transduction in activated thymocytes and subsequent DN3 cell differentiation mediated by the pMX-IRES-RFP vector

(A) The left panel indicates a viral transduction efficiency of 13.4%, whereas the right panels present the corresponding efficiency of DN3 cell differentiation to DP.

(B) Statistical analysis of viral transduction efficiency and DP cell differentiation from three independent experiments (with n = 4, 5, and 4 replicates in each) and shown as mean ± SEM.

Expected outcomes

We present a comprehensive in vitro protocol designed to facilitate the investigation of gene function in thymocyte development via retroviral transduction. The protocol includes the following key steps: (1) retroviral packaging, which necessitates a transfection efficiency of at least 80% for the core plasmid, as determined by the percentage of RFP-positive cells observed under microscopy, to ensure the production of high-titer virus; See Figure 1C as an example. We usually use 1-2×10⁶ TU/mL retroviral particles for the following transduction. (2) the isolation of murine DN3 thymocytes, which typically yields approximately 1×10⁶ cells per 4-6 week-old wild-type mouse, with a noted decrease in yield from older specimens; Please refer to Figure 2A for the gating strategy used for sorting. The results after DN cell enrichment and DN3 cell sorting are shown in Figure 2B. (3) the retroviral transduction of DN3 cells, which must be preceded by an essential 18-hour activation period on DLL4-coated plates, verified microscopically by the enlargement of cells, as insufficient activation significantly impairs infection efficiency; (4) in vitro differentiation, which requires the immediate replacement of the medium upon yellowing to maintain differentiation potential; and (5) flow cytometric analysis, which generally indicates a transduction efficiency from 10% to 40% across experimental replicates, with wild-type controls demonstrating greater than 80% differentiation into CD4⁺CD8⁺ (DP) cells. See Figure 3 as an example.

Limitations

This protocol is subject to three primary limitations: Firstly, DN3 cells must be activated prior to transduction to facilitate viral infection, which results in the analysis of gene function in activated cells rather than in their quiescent state. In addition, upon stimulation, a portion of DN3 cells differentiate into quiescent DP cells, thereby reducing the efficiency of retroviral infection. Therefore, when analyzing differentiation efficiency, it is essential to first gate on the RFP⁺ cells that have been successfully infected before proceeding with the analysis. Secondly, in vitro differentiation cultures can be maintained for a maximum of approximately five days, which poses challenges for subsequent experiments. Lastly, as reported, the TCR-α/β expression of SP cells in our in vitro DN3 differentiation assay is far less than that of ex vivo isolated SP cells.⁶ And in-vitro-differentiated SP cells fail to be activated by cell-surface CD3 cross-linking. Consequently, the applicability of our protocol is limited to the study of early thymocyte development (DN to DP) and is not suitable for extending to studies of late stages (DP to SP).

Troubleshooting

Problem 1

The generated retroviral particles are non-infectious.

Potential solution

Retroviral particles produced from Plat-E cells are non-infectious, as they lack essential viral genomic elements required for replication. Plat-E cells stably express the structural genes gag-pol and env (VSV-G) under continuous selection, enabling efficient packaging of exogenous genes while ensuring particle safety. To maintain stable long-term expression of these genes, Plat-E cells are routinely cultured in medium containing 10 μg/mL puromycin and 10 μg/mL blasticidin.

Problem 2

Low retroviral yield encounter during the viral packaging and harvesting stages.

Potential solution

Several factors contribute to suboptimal retroviral yields. Initially, it is essential to verify the viability and confluency of Plat-E packaging cells, as compromised cell conditions significantly reduce viral packaging efficiency, potentially necessitating the replacement of the cell line. In instances where Plat-E cells are unavailable, 293T cells may be utilized as a substitute; however, it is imperative to co-transfect these cells with both the core plasmid and the pCL-Eco packaging plasmid.^9,10 Secondly, ensuring uniform cell distribution and optimal density during transfection is crucial. Cells should be passaged at appropriate ratios based on their growth status from the previous day, gently resuspended to achieve monolayer homogeneity, and transfected when they reach approximately 80% confluency. Thirdly, the use of high-quality core plasmid DNA, with a concentration exceeding 1 μg/μL and free of endotoxins, is recommended to maximize transfection efficiency. Fourthly, the transfection complex should be added to the cell culture medium in a slow, dropwise manner while gently rocking the dish to ensure even distribution. Fifthly, strict adherence to post-transfection medium replacement protocols is necessary; the medium should be replaced at specified intervals using pre-warmed medium, which should be added carefully to avoid dislodging adherent cells. To minimize viral loss, it is recommended that the harvested supernatant be filtered using 0.45 μm PES membrane filters. Alternatively, virus stocks can be clarified through centrifugation at 3,000 × g for 5 minutes, with the supernatant retained for subsequent applications.

Problem 3

Low post-sort yield of DN3 thymocytes.

Potential solution

To optimize DN3 thymocyte yield, it is recommended to select mice aged 4–6 weeks, as older mice experience thymic atrophy, significantly reducing DN3 cell numbers. Furthermore, mechanical damage during single-cell preparation should be minimized—excessive grinding force on frosted glass slides or prolonged incubation during ACK lysis can compromise cell viability and recovery post-sorting. Allowing sorted cells to rest on ice for 60 minutes prior to centrifugation can help dissipate electrostatic repulsion between similarly charged cells, thereby facilitating efficient pelleting and addressing the issue of low post-sort yield of DN3 thymocytes.

Problem 4

Low retroviral transfection efficiency of DN3 thymocytes.

Potential solution

Several factors may contribute to a reduction in retroviral transduction efficiency. Firstly, inadequate pre-activation of DN3 cells can compromise infection, as retroviruses exclusively target proliferating cells. It is essential to plate cells at the recommended density of 5×10⁵ cells per well during DLL4/CXCL12 stimulation, as overcrowding can hinder activation. Secondly, freeze-thaw cycles of viral stocks should be avoided, as they degrade infectivity; thus, the use of freshly harvested supernatant is recommended. If storage is necessary, the viral supernatant should be aliquoted into small, single-use volumes prior to freezing. For subsequent experiments, only one aliquot is thawed for each use. Additionally, during spin transfection, it is important to maintain a cell density of ≤1×10⁶ cells per well and to set acceleration/deceleration ramping to level 3 to preserve cellular viability.

Problem 5

Reduced differentiation efficiency of DN3 thymocytes is observed following retroviral transduction.

Potential solution

Reduced differentiation efficiency of DN3 cells is primarily attributed to compromised cell viability due to prior manipulations or incorrect plating density. Beyond the risks to DN3 thymocyte viability mentioned before, other critical yet frequently overlooked factors include: (1) the accumulation of metabolic byproducts in acidified (yellowed) viral supernatant, which severely impairs both infectivity and the fitness of recipient cells. It is recommended to collect the viral supernatant at approximately 48 hours post-transfection, during the pre-acidification stage; and (2) positional stress experienced when culturing in perimeter wells during spin transfection, which necessitates use of central wells to maintain optimal cell conditions.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Linrong Lu (lu_linrong@zju.edu.cn).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Zejin Cui (cuizejin@renji.com).

Materials availability

This study did not generate new reagent.

Data and code availability

This study did not use any database and code.

Acknowledgments

This study was supported by the National Natural Science Foundation of China grants 32441096, U21A20199, 32141004, and 32350007 to L.L.; 82502114 to F.Z.; Innovative research team of high-level local universities in Shanghai SHSMU-ZLCX-20211600 to L.L.; and Internal Incubation Program of Renji Hospital RJTJ24-QN-076 to Z.C. We gratefully acknowledge Dou Liu, Dongliang Xu, and Pinpin Hou from the Core Facility of the Shanghai Immune Therapy Institute for their technical support. We thank Yanwei Li, Jiajia Wang, Yingying Huang, Chun Guo, Xin Shen, and Nan Zhou from the core facilities (Zhejiang University School of Medicine) for technical assistance in flow cytometric experiments. The graphical abstract was prepared with Figdraw.

Author contributions

F.Z. performed the experiment, Z.C. and F.Z. analyzed the data, Z.C. wrote the paper, and L.L. guided the study and reviewed the manuscript.

Declaration of interests

The authors declare no competing interests.