We are analyzing T cells in response to X antigen, and it seems there is an authoflourescent after compensation has been done. I was told that in order to elilminate autofluorescence i have to include an unused channel. However, I have used all channels, which is 8 color with FACS Canto II. I have also tried to apply unstimulated, and I thought I might loss my dim populations. Can I get any assistance?
Dead cells like to make autofluorescence. I would add live/dead staining, may be in the cost of one of the markers. I guess, one of them may be redundant.
Also I would make more strict gating on FSC and SSC as some bigger cells may be not lymphocytes, but something else, which may make such a trouble. And exclude doublets (however, it usually doesn`t influence so much).
First of all, if you can show the plots, it would be much easier for us to analyze.
Some sort of dead cells also cause increase in autofluorescen, that maybe removed by using live/dead stain.
Did you compensate your panel with beads earlier and now facing problem with cells? Compensation should be done with your cells of interest rather than beads.
You are looking at cells response to a stimulus by immunofluorescence. Are your cells alive or fixed ? If fixed, how are they fixed ? Formaldehyde ? Glutaraldehyde ? Ethanol ?
Is there any pH indicator in your culture medium such as Phenol Red ?
Do you use in your immuno mixture any reducing agent (Na, S2O5) in order to lower the flavin fluorescence ?
Olivier, I have fixed them with formaldehyde. No reducing agent and PH indicator too.
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