CellXViva Mouse T cell Activation Kit

Complete medium formulation for reference:

Protocol 1: Anti-Mouse CD3 Monoclonal Antibody Coating Method

1. Dilute NA/LE Rat anti-mouse CD3 Recombinant mAb into 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.

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

4. Remove 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, cells should be resuspended in complete medium at a concentration of (1-3)×10⁶ cells/mL. For a 24-well plate, a concentration of 1×10⁶ cells/mL is recommended.

6. Prepare fully supplemented T cell medium in a V-bottom tube, and add the following cytokines and antibodies to the cell suspension from step 6:

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

- 10ng/mL IL-2 Protein, Mouse

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

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

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

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

11. Centrifuge the cell suspension at 400 × g for 5 minutes, discard the supernatant. Resuspend the cells in 0.5 mL of 1% Bovine Serum Albumin (BSA) and count the cells. Assess cell viability, requiring >95% viability.

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 (S-R441) (S0B5215) according to the 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 PBS for flow cytometry analysis (requires >10,000 cells).