Due to logistics, I wonder if it would be possible to fix PBMCs and LPMCs overnight after surface stain before performing next day permeabilisation with foxp3 kit for transcription factors or BD kit for intracellular cytokines? If so what do you suggest I fix with: paraformaldehyde or the fixative that comes with the kit. Also would you fix for 1 hr then transfer to PBS for storage at 4 degrees overnight or leave in fixative overnight? I do use tandem dye such as pe-cy7 and pe-texas Red, would that be affected by this?
Is freezing cells after surface stain and fixation a better option?
Thanks.
Yes You can Fix, but it would affect the Pe-Cy7 stain index. It depends on the company providing the monoclonal and the molecule you target, e.g. anti-CD4 PEcy7 gating will not be much affected. but some low expressed molecules it would be So take care. needs experimental try first to see impact
I am not experienced with the kit that is coming from BD. However we are using a fix&perm kit that we have purchased from eBiosience where we use the PFA containing fixative that is coming with the kit and it makes no difference in FoxP3 staining if we fix the cells for one hour or overnight. I guess with the kit from BD it will be the same.
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