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Description
CellXViva Human T cell Activation KitProduct Specification Species Human Stability & Storage Store according to the instructions of each component Components Contain Reference dosage S Size L Size IL 2 Protein, Human 10 ng mL 5ug 10ug NA LE Mouse anti human CD3 mAb 5 g mL 1mg 5mg NA LE Mouse anti human CD28 mAb 5 g mL 1mg 5mg Protocol Protocol 1: anti Human CD3 mAb immobilized Method 1. Dilute NA LE Mouse anti Human CD3 mAb into a final concentration of 5g mL in PBS. 2. Immobilize NA LE
Product Specification
| Species | Human |
| Stability & Storage | Store according to the instructions of each component |
Components
Contain
|
Reference dosage
|
S Size
|
L Size |
IL-2 Protein, Human |
10 ng/mL |
5ug |
10ug |
5 µg/mL |
1mg |
5mg |
|
5 µg/mL |
1mg |
5mg |
Protocol
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).
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