T cells are central to the immune system, playing a critical role in combating infections and tumors. Due to their immunological similarities to humans, mouse spleen T cells serve as a vital model for studying T cell function, immune mechanisms, and developing immunotherapies (such as CAR-T). This protocol addresses issues like low efficiency in in vitro activation and expansion, as well as poor cell condition, providing standardized technical support for related research.

I. Experimental Principle

The mouse spleen, a vital peripheral immune organ, is rich in T lymphocytes (comprising approximately 40%-50% of total splenic lymphocytes), including CD4⁺ helper T cells, CD8⁺ cytotoxic T cells, and other subsets. This protocol employs mechanical dissociation to obtain a single-cell suspension from the spleen. Following enrichment of T cells via immunomagnetic bead sorting, specific activation signals are provided using anti-CD3/CD28 monoclonal antibodies. Combined with cytokines (e.g., IL-2) to sustain proliferative activity, this approach achieves highly efficient in vitro expansion of T cells. Strict control of culture conditions during expansion prevents heterocellular contamination and T cell functional exhaustion, ensuring the purity, activity, and stable immunological function of the expanded cells.

II. Materials and Reagents

1. Experimental Animals

6-8 weeks-old C57BL/6 or BALB/c mice (strain selected based on experimental requirements), supplied by a certified animal research center and housed in SPF-grade animal facilities.

2. Core Reagents

1) Basic Medium: RPMI-1640 medium (containing L-glutamine, 25 mM HEPES), supplemented with 10% fetal bovine serum (FBS, heat-inactivated), 100 U/mL penicillin, and 100 μg/mL streptomycin. Filter-sterilized and stored at 4°C; equilibrate to room temperature prior to use.

2) Cell Sorting Reagents: Antibody-conjugated magnetic beads.

3) Activation reagents: Anti-mouse CD3ε monoclonal antibody, anti-mouse CD28 monoclonal antibody.

4) Cytokines: Recombinant mouse IL-2, stored frozen at -80°C. Dilute in basal medium prior to use.

5) Auxiliary reagents: ACK Lysing Buffer, PBS buffer (pH 7.4), 0.25% trypsin-EDTA solution, trypan blue staining solution (0.4%), flow cytometry antibodies (CD3-PE, CD4-FITC, CD8-APC for purity assessment).

3. Experimental Equipment

Biosafety Cabinet, CO₂ incubator (5% CO₂, 37°C, saturated humidity), Centrifuge, Flow cytometer, Inverted microscope, Cell counting chamber, Sterile centrifuge tubes (15 mL/50 mL), Sterile culture flasks (25 cm²/75 cm²), Sterile grinding rods, 200-mesh cell strainer, Magnetic bead sorting column, Magnetic stand, Pipettes and sterile pipette tips.

III. Experimental Procedure

1. Mouse Spleen Harvesting

1) Euthanize mice by cervical dislocation and immerse in 75% ethanol for 3–5 min.

2) Fix the mouse in the supine position. Incise the abdominal skin and peritoneum to expose the spleen. Remove the spleen with forceps and place it in a culture dish containing 5 mL of pre-chilled PBS. Remove surface connective tissue and fat.

2. Preparation of Mouse Spleen Single-Cell Suspension

1) Gently grind the spleen using a sterile grinding rod. Filter the suspension through a 200-mesh cell strainer into a 15 mL centrifuge tube. Rinse the strainer repeatedly with PBS and collect the filtered cell suspension, avoiding vigorous grinding that could cause cell rupture.

2) Centrifuge at 1500 rpm for 5 min (4°C). Discard the supernatant. Add 3 mL ACK red blood cell lysis buffer and incubate at room temperature for 3-5 min, gently inverting the tube 2-3 times to ensure complete red blood cell lysis.

3) Add 10 mL pre-chilled PBS to terminate lysis. Centrifuge at 1500 rpm for 5 min (4°C). Discard the supernatant. Wash cells twice with PBS (centrifuge at 1500 rpm for 5 min each wash) to remove residual lysis buffer and cell debris.

4) After the final centrifugation, discard the supernatant. Resuspend cells in 5 mL of basal medium. Take a small volume of the cell suspension for trypan blue staining and count live cells using a hemocytometer (viability ≥ 90%). Reserve the remaining cells for subsequent sorting.

3. Magnetic Bead Sorting for T Cell Enrichment

1) Cell Counting: Resuspend cells in buffer and adjust concentration to 1×10⁷–1×10⁸ cells/mL.

2) Magnetic Bead Incubation: Add CD3 antibody magnetic beads according to the reagent manual ratio (typically 10 μL beads/1×10⁷ cells). Incubate at room temperature in the dark for 15–20 minutes, gently inverting the tube 2–3 times during incubation.

3) Magnetic separation: Place the tube on a magnetic stand and allow it to stand at room temperature for 5 minutes. Aspirate and discard the supernatant (cells not bound to magnetic beads). Add wash buffer and wash twice, allowing each wash to stand for 3 minutes before discarding the supernatant.

4) Elute target cells: Remove the tube from the magnetic stand, add an appropriate volume of base buffer, gently resuspend cells by pipetting, and collect the liquid containing CD3+ T cells.

5) Purity Verification: A small amount of sorted cells is stained with CD3 fluorescent antibody and analyzed by flow cytometry to ensure purity ≥ 90%.

4. T Cell Activation Stimulation

1) Coating Culture Plates: One day prior to use, coat cell culture plates (e.g., 6-well plates) with an appropriate amount of anti-CD3ε antibody solution (typically 1-5 μg/mL in PBS) overnight at 4°C or for 2 hours at 37°C. After incubation, aspirate the coating solution and wash once with PBS to remove unbound antibody.

2) Seeding and Stimulation: Seed spleen cell suspensions at a density of 1-2 × 10⁶ cells/mL into the antibody-coated culture plates.

3) Add Stimulatory Factors: Add soluble anti-CD28 antibody (commonly at 1-2 μg/mL) and recombinant mouse IL-2 (recommended starting concentration 20-50 U/mL) to the medium. Gently mix and incubate at 37°C in a 5% CO₂ saturated humidity incubator.

5. T Cell Expansion and Maintenance

1) Observation and Medium Change: After 24-48 hours of activation, cell aggregates will form and increase in volume, indicating successful activation. Typically, a partial or complete medium change is performed after 48-72 hours of culture.

2) Nutrient and Factor Supplementation: During medium exchange, carefully aspirate half of the old medium and add an equal volume of fresh complete medium containing IL-2 (50-100 U/mL). Thereafter, perform medium changes or passages every 2–3 days based on cell density and medium color changes (e.g., yellowing), maintaining cell density between 0.5–2 × 10⁶ cells/mL to prevent apoptosis caused by overcrowding.

3) Expansion Period: Under sustained stimulation, T cells typically proliferate for 5–10 days, increasing in number by tens to hundreds of times.

6. Cell Phenotype and Functional Identification

1) Viability Assessment: Trypan blue staining and counting; Viability should be ≥ 85%.

2) Purity Identification: Flow cytometric analysis of CD3⁺ T cell proportion and distribution of CD4⁺ T cell and CD8⁺ T cell subsets.

3) Functional Identification: Verify the immune function of expanded T cells by detecting cytokine secretion capacity (IFN-γ, IL-2) via ELISPOT or assessing CD8⁺ T cell-mediated cytotoxicity against target cells through cytotoxicity assays (e.g., LDH release assay).

IV.  Key Optimization Points

1. Antibody Concentration Optimization: Excessively high concentrations of anti-CD3/CD28 antibodies may lead to T cell overactivation and exhaustion, while insufficient concentrations result in inadequate activation. Conduct preliminary experiments with gradient concentrations (CD3: 2, 5, 10 μg/mL; CD28: 1, 2, 5 μg/mL) to identify optimal activation concentrations.

2. IL-2 Concentration Adjustment: IL-2 is a key cytokine for T cell proliferation. Low concentration slows proliferation, while high concentration may induce regulatory T cell (Treg) proliferation. Adjust the concentration (10-50 U/mL) according to experimental requirements. For effector T cell enrichment, use 30-50 U/mL.

3. Cell Seeding Density: Initial seeding density that is too low impairs cell-cell interactions, reducing activation efficiency; excessively high density may cause nutrient depletion and metabolic waste accumulation. Maintain an initial density of 1×10⁶ cells/mL, and sustain 5×10⁵–1×10⁶ cells/mL during expansion.

4. Culture Environment Control: Strictly maintain CO₂ concentration at 5%, with saturated humidity and minimal temperature fluctuations (37 ± 0.5°C). Use freshly prepared medium and select FBS from batches with stable lot numbers and free of mycoplasma contamination to minimize risk of contaminating cell lines.

V. Experimental Precautions

1. Sterile technique must be strictly adhered to throughout the experiment. All equipment and reagents must be sterilized. The laminar flow hood should undergo 30 minutes of UV irradiation prior to use to prevent bacterial and fungal contamination.

2. Spleen tissue harvesting and cell isolation must be performed rapidly. Cells should be kept on ice or at 4°C throughout to minimize loss of viability. Grinding should be performed with moderate force to avoid mechanical damage causing cell rupture.

3. During magnetic bead sorting, prepare sorting buffer fresh for immediate use to prevent EDTA precipitation from affecting sorting efficiency. Keep sorting columns moist to prevent column bed desiccation.

4. Regularly monitor cell status during amplification. If excessive cell aggregation, cloudy medium, or unusual odor occurs, contamination may be present. Discard contaminated samples immediately and investigate the source of contamination.

5. Expanded T cells should be used promptly for experiments. For short-term storage, they may be kept at 4°C for up to 24 hours or suspended in cryoprotectant containing 10% DMSO and stored at -80°C using a rapid freeze-thaw protocol to prevent functional decline from prolonged room-temperature exposure.

VI.  Yeasen HiActi™ Cytokines

Recombinant proteins have been widely applied in core fields such as stem cell and organoid culture, recombinant protein therapeutics, CAR-T cell therapy, and antibody drugs. With the rapid development of the biopharmaceutical industry, the recombinant protein market has experienced explosive growth, and demand for high-end raw materials has risen year by year. To precisely meet the continuously upgrading application needs in both research and industrial settings, and to address key pain points such as low protein activity and insufficient batch-to-batch stability, Yeasen Biotech has leveraged its years of R&D, production experience, and technological accumulation to build an innovative recombinant protein expression and purification platform. This platform focuses on providing high-activity recombinant protein products. Leveraging its proprietary expression and purification platform, Yeasen Biotech has developed a series of HiActi™ cytokines—including IL-2, IL-7, and IL-15—for mouse splenic T cell culture. These products undergo rigorous quality control and cellular function validation to ensure high activity, purity, stability, and low endotoxin levels, helping you achieve optimal experimental results.

Commonly Used Cytokines for Mouse Spleen T Cell Culture

Cytokines	Core Function
IL-2	Promotes T-cell clonal expansion, enhances cellular activity, inhibits apoptosis after activation, and maintains long-term in vitro culture
IL-7	Maintains T cell survival and homeostasis, supports primary T cell culture, reduces exhaustion, and promotes memory T cell formation
IL-15	Potently promotes CD8+T cell proliferation and survival, maintains cytotoxicity, and stabilizes memory T cell phenotype
IL-12	Induces differentiation of naive T cells toward the Th1 subset, enhances IFN-γ secretion, and boosts cellular immune responses
IL-4	Promotes T cell differentiation toward the Th2 subset and enhances humoral immunity-related functions
IL-21	Enhances CD8+T cell cytotoxic function, inhibits T cell exhaustion, and modulates immune cell activity
TNF-α	Low concentrations synergistically promote T cell activation and proliferation (high concentrations inhibit)

Yeasen Product Data

Bioactivity of Mouse IL-2 

Figure 1. Measured in a cell proliferation assay using CTLL-2 mouse cytotoxic T cells. The EC₅₀ for this effect is 0.01–0.1 ng/mL.

Bioactivity of Mouse IL-7 

Figure 2. Measured in a cell proliferation assay using PHA-activated human peripheral blood lymphocytes (PBL). The EC₅₀ for this effect is 0.01–0.2 ng/mL.

Bioactivity of Mouse IL-12

Figure 3. Measured in a cell proliferation assay using PHA-activated mouse splenocytes. The EC₅₀ for this effect is 0.5-4 pg/mL.

Ordering information

Product Name	Cat. No.	Specification
Recombinant Mouse IL-2 Protein	90142ES	5μg/50μg/100μg/500μg
Recombinant Mouse IL-4 Protein	90144ES	5μg/50μg/100μg/500μg
Recombinant Mouse IL-7 Protein	90147ES	2μg/10μg/50μg/100μg/500μg
Recombinant Mouse IL-12 Protein	90193ES	10μg/50μg
Recombinant Mouse IL-15 Protein, His Tag	90218ES	10μg/20μg/50μg/100μg
Recombinant Mouse IL-21 Protein (HEK293)	90233ES	10μg/50μg/100μg/500μg
Recombinant Mouse TNF-α/TNFSF2	90621ES	5μg/20μg/50μg/100μg/1mg