CellXViva Mouse T cell Activation Kit

Protocol 1: anti-Mouse CD3 mAb immobilized Method

1. Mouse T Cell Growth Medium Nutritional System:

• RPMI 1640 Medium, ATCC Modified (with L-glutamine, sodium pyruvate, and HEPES) (abs9468)
• 12% Premium Fetal Bovine Serum (abs972)
• 1% Antibiotic-Antimycotic (abs9244)
• Cell Freezing: Serum-Free Cryopreservation Medium for Immune Cells (abs9999)

Note: The above medium system is for reference only.

2. Dilute NA/LE Rat anti-mouse CD3 Recombinant mAb into a final concentration of 5µg/mL in PBS.

3. Immobilize NA/LE Rat anti-mouse CD3 Recombinant mAb by adding 1mL/well diluted mAb into 24-well plate.

4. Incubate at 37 °C and 5% CO₂ for 2h, or 4 °C overnight.

5. Drain NA/LE Rat anti-mouse CD3 Recombinant mAb from 24-well plate.

6. Preparation of single-cell suspension from mouse spleen. For T cell activation, the cells should be resuspended in complete medium at (1-3)×10⁶cells/mL. 1×10⁶is recommended for 24-well plate.

7. Prepare fully supplemented T cell medium in V-shaped tube by adding cytokines and antibodies to the cell suspension from step 5 as follows:

•  5µg/mL NA/LE Syrian Hamster anti-mouse CD28 mAb
•  10ng/mL IL-2 Protein, Mouse

8. Transfer the desired amount of cells to a suitable cell culture plate to a final density of (1-1.5)×10⁶ cells/mL/cm².

9. Incubate at 37 °C and 5% CO₂ for 2 days.

10.  After 2 days of cultivation, examine for cell healthiness and record image.

11.  Centrifuge cell suspension at 400×g for 5 min. Discard medium supernatant. Wash cell pellet in PBS.

12.  Centrifuge cell suspension at 400×g for 5 minutes. Discard supernatant. Resuspend cell in 0.5mL 1% BSA. Determine cell number. Examine for cell viability, >95% viability is required.

13.  Block 1×10⁶cells in 10µg Mouse IgG antibody. Incubate 30min in ice bath.

14.  Add FITC Rat Anti-Mouse CD25 Antibody (S-R441) (S0B5215) guided by the manufacture manual, incubate 30min at 4 °C.

15.  Centrifuge cell suspension at 400×g for 5 minutes. Wash cell pellet in 1% BSA containing PBS twice.

16.  Resuspend cell pellet in a 200µl PBS for analysis by flow cytometry (>10000 cells required).