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