When doing Flow Cytometry, I used B16 cells as negative control of IFN-gamma and Granzyme B. But apparently, the sizes of B16 and lymphocytes are so different. Is this allowed? Or, I can only use the same lymphocytes as negative group?
I agree with Antonio, for an IFNg negative control, use your lymphocyte cells stained with all the other antibodies you are using (not IFNg), and add in an isotype control for IFNg. This sort of control is stimilar to fluorescence minus one (FMO) controls. If you google that, BD and ebio give explanations of why FMO's are important.
Hi Yang, there is so much variability in different cells as well as stimuli and during the staining process. Using the same type of cells and staining under the same condition as well as flow acquisition of the samples will be great and will guarantee a successful data analysis process.
I agree with Elvis Kidzeru . firstly, different cells have different forward and side light-scatter characteristics,which will affect on the instrument setting; Secondly, both IFNg and Granzyme B perform intracellular staining ,usually positive cells are very small population in this case, so performer must set strict controls,using the same cells as control for single color staining and fluorescence minus one (FMO) controlsbased on the same cells for multicolor staining .
I agree with the above, and suggest
1. in addition to autofluorescence (which could be adjusted prior to analysis), cell have distinct, unpredictable properties of non-specifically binding Abs, such that theoretically each Ab and each cell type is a "unique pair".
2. positive control are less critical, as long as the particular staining is prominent.
3. The best controls in my opinion are those using the soluble antigen. For instance, peptide-raised antiserum should include the peptide+Ab pre-mix as a control. In your case, I suspect monoclonals, IFNg should be easy to add, granzyme B may be more difficult to come by.
Good luck - Klaus
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