The Mouse T Cell Activation Kit (Antibody Coating Method) is specifically designed for primary resting T cells isolated from mouse spleen, lymph nodes, and peripheral blood. The kit efficiently activates various T cell subsets, including CD4⁺, CD8⁺, and Treg cells, and is compatible with conventional cell culture plate-based solid-phase activation systems.

Activation Principle: Complete in vitro T cell activation relies on the synergistic coordination of dual signals. This kit employs a solid-phase antibody coating strategy in which antibodies are immobilized on the culture plate surface to provide sustained and uniform stimulation. This approach overcomes the limitations of soluble antibody stimulation—including weak activation signals and heterogeneous activation—thereby ensuring stable T cell activation, normal proliferation, and differentiation. The core principles are as follows:

1. First Signal (Antigen Recognition Signal): High-purity anti-mouse CD3 monoclonal antibody is immobilized on the bottom of the culture plate via solid-phase coating, enabling sustained and stable binding to the TCR-CD3 complex on the T cell surface. This efficiently initiates the core antigen recognition signaling pathway, awakening resting T cells to undergo initial activation with persistent and uniform signal stimulation.

2. Second Signal (Co-stimulatory Signal): Co-coated anti-mouse CD28 specific antibody delivers a critical co-stimulatory signal that effectively overcomes T cell anergy, continuously activates downstream NF-κB, MAPK, and other core signaling pathways, and promotes T cell activation and survival—providing the necessary conditions for subsequent proliferation and subset differentiation.

3. Proliferation Auxiliary Signal: The kit is supplemented with high-activity recombinant mouse IL-2 cytokine, which provides specific proliferation signals to activated T cells. This effectively promotes large-scale T cell expansion, maintains cellular physiological activity and differentiation stability, prevents post-activation apoptosis and proliferation arrest, and supports long-term culture and differentiation experiments.

Product Information

Product Components

Component Information

Reconstitution Method (Mouse IL-2)

Before opening the vial, centrifuge briefly to bring the contents to the bottom. Reconstitute with sterile distilled water or PBS to a final concentration of 0.1–1.0 mg/mL. Further dilution may be performed according to downstream experimental requirements. For long-term storage, it is recommended to add a carrier protein (e.g., 0.1% BSA) to the dilution buffer. Aliquot into single-use volumes and store at –80°C. Avoid repeated freeze-thaw cycles.

Mouse T Cell Activation Protocol

Complete Medium Formulation (for reference):

Anti-mouse CD3 Monoclonal Antibody Immobilization Method:

1. Dilute NA/LE Rat anti-mouse CD3 Recombinant mAb in PBS to a final concentration of 10 μg/mL.

2. Add the diluted NA/LE Rat anti-mouse CD3 Recombinant mAb to a 24-well plate at 1 mL per well for coating.

3. Incubate at 37°C with 5% CO₂ for 2 hours, or incubate overnight at 4°C.

4. Aspirate the NA/LE Rat anti-mouse CD3 Recombinant mAb from the 24-well plate.

5. Prepare a single-cell suspension from mouse spleen. For T cell activation, resuspend cells in complete medium at a concentration of (1–3)×10⁶ cells/mL. For a 24-well plate, the recommended concentration is 1×10⁶ cells/mL.

6. Prepare complete T cell medium supplemented with the following cytokines and antibodies in a V-bottom tube, and add them to the cell suspension from Step 5:

    —2 μg/mL NA/LE Syrian Hamster anti-mouse CD28 mAb

    —10 ng/mL IL-2 Protein, Mouse

7. Transfer the required volume of cells to an appropriate cell culture plate at a final density of (1–1.5)×10⁶ cells/mL/cm².

8. Incubate at 37°C with 5% CO₂ for 2 days.

9. After 2 days of culture, examine cell health and record images.

10. Centrifuge the cell suspension at 400×g for 5 minutes and discard the supernatant. Wash the cell pellet with PBS.

11. Centrifuge the cell suspension at 400×g for 5 minutes and discard the supernatant. Resuspend the cells in 0.5 mL of 1% bovine serum albumin (BSA) and count the cells. Assess cell viability; viability should be >95%.

12. Block 1×10⁶ cells with 10 μg of mouse IgG antibody. Incubate on ice for 30 minutes.

13. Add FITC Rat anti-mouse CD25 antibody (clone S-R441) (S0B5215) according to the manufacturer's instructions and incubate at 4°C for 30 minutes.

14. Centrifuge the cell suspension at 400×g for 5 minutes and wash the cell pellet twice with PBS containing 1% BSA.

15. Resuspend the cell pellet in 200 μL of PBS for flow cytometry analysis (>10,000 cells required).

Product Properties

Features

• High-efficiency activation, high-activity expansion, low endotoxin, and user-friendly operation.

Applications

Case Study 1: Flow Cytometry Analysis

Flow cytometry analysis was performed to evaluate the activation efficacy of the Mouse T Cell Activation Kit. Mouse splenocytes were cultured under three conditions: unstimulated (Unable), negative control, and immobilized anti-CD3 mAb treatment. CD25 surface expression was measured as a marker of T cell activation.

Figure 1: Flow cytometry analysis of CD25 expression on mouse T cells under different conditions. Compared with the unstimulated (2.18%) and negative control (1.92%) groups, T cells activated with the immobilized anti-CD3 mAb showed significantly increased CD25 expression (85.1%), demonstrating effective T cell activation using this kit.

Storage

Store at –25°C to –15°C. The shelf life is 1 year from the date of receipt. Avoid repeated freeze-thaw cycles. Centrifuge briefly before opening the vial. It is recommended to aliquot into single-use volumes to prevent repeated freeze-thaw cycles.

Documents:

Safety Data Sheet

92675_MSDS_HB260824

Manual

92675_Manual_HB20260821