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Description
CellXViva Mouse T cell Activation KitProduct Specification Species Mouse Stability & Storage Store according to the instructions of each component Components Contain Reference dosage S Size L Size IL 2 Protein, Mouse 10 ng mL 5ug 10ug NA LE Rat anti mouse CD3 Recombinant mAb 5 g mL 1mg 5mg NA LE Syrian Hamster anti mouse CD28 mAb 5 g mL 1mg 5mg Protocol Protocol 1: anti Mouse CD3 mAb immobilized Method 1. Mouse T Cell Growth Medium Nutritional System: RPMI 1640 Medium, ATCC Modified
Product Specification
| Species | Mouse |
| Stability & Storage | Store according to the instructions of each component |
Components
Contain |
Reference dosage |
S Size |
L Size |
IL-2 Protein, Mouse |
10 ng/mL |
5ug |
10ug |
NA/LE Rat anti-mouse CD3 Recombinant mAb |
5 µg/mL |
1mg |
5mg |
NA/LE Syrian Hamster anti-mouse CD28 mAb |
5 µg/mL |
1mg |
5mg |
Protocol
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).
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