Hi flow users/cell masters,
I'm just wondering if anyone here has experience with staining cytoplasmic cytokines after staining extracellular proteins. So basically what I did in my protocol was staining extracellular markers using PE-conjugated and APC/Cy7-conjugated antibodies, then fix/perm my samples using the 1X fix/perm buffer eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set before staining cytoplasmic cytokine using antibodies resuspended in 1X perm buffer.
I didn't see any APC/Cy7+ or PE+ populations in flow, which is weird coz the markers are supposed to be highly expressed. I saw some posts on Reddit that APC/Cy7 is not stable, but I don't know if PE is unstable either. Also, does anybody know if eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set contains methanol? I couldn't find the info anywhere, if it is, it makes sense then... Since PE and APC tandems are not methanol-resistant.
Thanks in advance to anyone who's going to answer this :)
You do appear to mention what you are staining for and why you are using this reagent? I find the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set is very effective at permeabilising cells for nuclear staining. However, I believe it contains formaldehyde and methanol.
I would suggest you try a detergent based permeabilisation - I find fixation with paraformaldehyde and permeabilisation with 0.1 0.5% Saponin works well for intracellular staining. You can commercial reagents which are QC'd eg BD bioscience or make your own.
best wishes
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