Protocol 1: anti-Human CD3 mAb immobilized Method

1. Dilute NA/LE Mouse anti-Human CD3 mAb into a final concentration of 5µg/mL in PBS.

2. Immobilize NA/LE Mouse anti-Human CD3 mAb by adding 1mL/well diluted mAb into 24-well plate.

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

4. Drain NA/LE Mouse anti-Human CD3 mAb from 24-well plate.

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

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

l 5µg/mL NA/LE Mouse anti-Human CD28 mAb

l 10ng/mL IL-2 Protein, Human

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

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

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

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

11. 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.

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

13. Add Brilliant Violet 421TM anti-Human CD25 antibody (302630) and Alexa Fluor® 488 anti-human CD69 Antibody (310916) guided by the manufacture manual, incubate 30min at 4 °C.

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

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

Protocol 2: anti-Human CD3 mAb mobilized Method

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

2. Prepare fully supplemented T cell medium in V-shaped tube by adding cytokines and antibodies to the cell suspension from step 2 as follows:

l 5µg/mL NA/LE Mouse anti-Human CD3 mAb

l 5µg/mL NA/LE Mouse anti-Human CD28 mAb

l 10ng/mL IL-2 Protein, Human

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

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

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

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

7. 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.

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

9. Add Brilliant Violet 421TM anti-Human CD25 antibody (302630) and Alexa Fluor® 488 anti-human CD69 Antibody (310916) guided by the manufacture manual, incubate 30min at 4 °C.

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

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