I've tried to induce inf-g with frozen cells in the past, I find that although the production occurs, the external immunophenotyping isn't optimal for several molecules. CD4 seems to suffer from the thawing, as does CD56. CD8 and 3 don't seem to change as much, neither do CD19/20. I was also able to detect CD69 production on the same cells but only with the strongest activators. Also, even my most positive controls yield lower production of CD69 and inf-g on frozen cells than their non-frozen controls. Frozen are more likely to have higher background. But inf-g signal is usually in the 3rd log (depending on your fluorochrome, I would recommend BD PE or Caltag RPE conjugates) Maciej
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