Hi, guys
I have had big problem with Jurkat cell activation.
Protocol is as follow:
1. Seed jurkat at 1X 10^5 cell/ml and incubate for O/N
2. Treat jurkat with PHA(sigma, L1668-5MG) at 1, 10, and 50 μg/mL, and PMA (sigma, P1585-1MG) at 50, 100 ng/mL for 24, and 48 hours
3. Collect cell culture supernatants
4. IL-2 ELISA (abcam, ab270883)
But, IL-2 was not released from jurkat stimulated by PMA/PHA.
Instead of PHA, Jurkat was treated with PMA/ionomycin (invivogen), however, IL-2 secretion was not shown (PMA at 50 ng/ml, Ionomycin at 1 ug/ml).
I doubled the cell density, but it didn’t work out.
What's the matter with my experiments?
Is the cell in bad condition? Or Should I change the ELSIA kit?
Thank you
recently, I tried PMA/ionomycin and it didn't produce IL-2, （PMA25ng/ml,50ng/ml and 100ng/ml stimulat 12H )
You may be missing stimulation via CD28. We co-stimulate Jurkat T cells with anti-CD3 antibody OKT3 and an anti-CD28 antibody and get a good, ELISA-measured IL-2 response. It also works when we express recombinant murine-derived TCRs in Jurkat cells and stimulate with a murine TCR-specific antibody (H57) or the actual antigen presented by MHC, but again only when adding an anti-CD28 antibody. The anti-CD28 antibdy by itself does not induce IL-2 production.
- Badges
- Science topic