Cytokine Production


1. Coat plates if needed for overnight, 4°C. Use 96 well plates, flat bottom.

2. Count cells, and resuspend cells in 10%FBS/RPMI (for splenocytes, 10^7 cells/ml do each well will have 10^6 cells).

3. Add 100 μl of medium or cytokines etc. into the well.
For anti-CD3, coat wells with anti-CD3 (0.5-10 mg/ml for O/N and suck off before use).

For other reagents:

Stock Concentration:

Anti-mIgM/Fab 1.3 mg/ml
Anti-CD40 1 mg/ml
IL-4 1 mg/ml
LPS 1 mg/ml
PMA 20 μg/ml
Ionomycin 1 mg/m

Final Concentration:

Anti-mIgM/Fab 5 mg/ml
Anti-CD40 1 μg/ml
IL-4 1 μg/ml
LPS 1 μg/ml
PMA 20 ng/ml
Ionomycin 1 μg/ml l

4. Add 100 μl cells to each well so final volume in each well is 200 μl.

5. Incubate for 4-24 hours in CO2 incubator.

6. Take the supernatant out and perform ELISA (see ELISA protocol for detail)