Hi everyone,
I am staining leukocytes isolated from mouse kidneys in flow cytometry. I stimulated the cells with PMA/Ionomycin + BFA for 4 hrs in T-cell medium (RPMI + Pen/Strep + 10 % FCS + 50 uM beta-mercaptoethanol) and stained them for surface markers, T-cell transcription factors and IFNg and IL17A.
When I plot the cytokine production (IFNg-BV711 vs IL17A-BV650) in CD4+ T cells, I noticed that beside my single-positive populations, there are events on a somewhat straight diagonal line that seem to be double-positive. There are some other events that are double-positive that are more scattered around, which is why I think the events on the diagonal could be a technical artifact (see attached plot).
I am also attaching my FMOs for IFNg-BV711 and IL17A-BV650 where these events are not present.
I'd highly appreciate your thoughts on this.
Thanks a lot,
Jasper
It is autofluorescence. You need to block the unspecific binding of your antibodies with BSA or FBS and to use filtered FACS solution.
Thanks a lot for your answer Libia.
We do incubate our cells with the BD Fc Block (anti-CD16 and anti-CD32) before the surface marker staining. After surface staining we fix and permeabilize the cells with the Foxp3 transcription factor kit, wash twice with the wash buffer from the kit and then resuspend the cells in the antibody mix that stains for intracellular antigens. So we do not have a blocking step before the intracellular staining. I‘m assuming you would recommend this then? Would you mind sharing what concentration of FBS you would suggest and for how long you would block?
Thanks!
Jasper
That might be autofluorescent dead cells. You should add a viability stain so that you can exclude the dead cells. There are many amine binding alternative to chose from. You stain with the viability stain before continuing with surface staining, fixation and intracellular staining. ThermoFisher have many fixable viability stains from eBiosciences, Biolegend have their Zombie Fixable viability kits and BD Biosciences have some too.
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