I am doing peptide stimulation of spleen T cell and IFNr staining.
Here's my protocol, I thaw the spleen cells (I have already done tetramer staining so I am sure around 10% of the CD8 T cell should respond to the stimulation) one day before the stimulation and the second morning I stimulate with 10ug/ml peptide + 1ug/ml BFA for 6h and I also included PMA+ionomycin+BFA as a positive control and BFA only for a negative control. After that I did IFNr and CD8 staining. I got a clear IFNr population but the peptide and PMA+iono didn't show any increase. My questions are
1. I check the viability of the frozen spleen cell, It's around 40%. Did I have to use fresh isolated spleen cells?
2. Even without stimulation I still got IFNr stimulation, is it normal?
I know of a few examples where cryopreservation of PBMCs has been found to result in higher basal/spontaneous production of IFNg (e.g., see Diabetes. 2007 Mar;56(3):613-21.), so I imagine that an analogous situation might occur with (mouse?) splenocytes.
That being said, you should still expect to see an increase over background, particularly with PMA+Iono stimulation. What was the % of IFNg+ cells under your various stimulation conditions?
Thanks, Adeeb.
Actually I did virus infection first and then collect the splenocytes on Day13. Maybe that's reason why I got high basal level IFNg positive.
I just checked my staining, IFNg/CD8 percentage are 7%, 7.1% and 7.7% in control treatment, peptide stimulation and PMA+iono treatment respectively.
Jacky, you may try to reduce the peptide concentration up to 1 ug/ml or less, you may try to activate cell earlier after thaw; you can check IFN-g and IL-2 protein in supernatant. you should try to reduce your IFNg background first by media modification. you should try to use other mitogens (ConA). freshly isolated cells is always good choice! Mitogen response should be always positive, otherwise you doing something wrong. Good luck! IB
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology