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% CO??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)×10?cells/mL. 1×10?is 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)×10??cells/mL/cm2.
8.?Incubate at 37 °C and 5% CO??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 1×10?cells 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)×10?cells/mL. 1×10?is 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)×10??cells/mL/cm2.
4.?Incubate at 37 °C and 5% CO??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 1×10?cells 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).
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