Mitogen titration

ZellNet QuestionsCategory: QuestionsMitogen titration
Jessica Flechtner asked 3 years ago

I am interested in titering mitogen in the IFN-gamma T cell fluorospot assay to achieve a low frequency of responding cells. Has anyone had success titering non-specific stimuli to consistently yield fewer than 100 SFC/million from human T cells?

1 Answers
Sylvia Janetzki Staff answered 3 years ago

Hello Jessica; decreasing the concentration of mitogen will result in less and less well defined spots. It is not recommended. A better approach is to use less cells per well. But then – it is impossible to get to <100 spots/Million cells. For example, if you add only 50,000 cells per well, you will still get more than 5 spots per well, what is the same as 100/Million. Anyhow, 30-50k cells per well should give you a relatively low spot number, which should give no problems in evaluating/counting them.