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

Fresh cells give a better response in this type of assays. Tit rate the peptide concentrations for optimum stimulation and do a kinetic experiments. Spontaneous IFN-gamma producers are present in some experiments [may be due to invivo activation]. The kinetics do differ for ifn-gamma positivity [cells] for mitogen and peptide responses. lalitha kabilan