I am trying to put together a panel to simultaneously stain for cell surface markers with Hoechst, p-RB and p-H3S10, as well as activation markers for CD8 T cells including CD69, CD25 and CD44, as well as CD8.
I will need to fix in 2% PFA and perm in ice cold -20oC 90% MeOH. I know this protocol works well for the cell cycle stain, but I am worried about how the cell surface stain will be effected by this treatment
Currently I have p-RB in af488 and p-H3S10 on PE. I've been able to put together a panel based on the antibodies I have available, and includes CD69-APC, CD25-PE-Cy7, CD44-PerCP, CD8-Texas Red.
However, while this is fine regarding spectral overlap, I have recently discovered that certain antibody-flurophore combinations do not survive ice cold 90% MeOH, with CD69-APC being one of them. On top of that, I'm wondering if using tandemn dyes in -20 MeOH for even 15 minutes would be enough to degrade them, making them appear in different channels.
Ideally I wouldn't want to use a detergent to perm, as there may be a chance later down the line that I can use this method to sort cells for MS analysis, and detergents would contaminate MS findings.
Has anyone else managed to overcome similar issues of designing a panel with surface and intracellular staining using MeOH to perm?
If so, are there particular fluorophors or clones you recommend?
Or would it be best to abandon the combined stain and just keep them as separate panels?
Dear David Alexander Lewis
I just briefly wanted to email you and send you some considerations for your experiment. I have done a pretty similar experiment albeit with Ki67 as cell cycle marker. Speaking from experience, I have always kept my activation and my cell cycle stain separate as the methanol permeabilization just doesn't work well with tandem dyes for extracellular staining prior to the fixation/ permeabilization step.
On a second note, even though you're right the methanol fixation attenuates the APC signal, it does not totally abolish it and you can use the dye, if you combine it with an antigen that is abundantly expressed (usually CD4 or CD8).
I have tried and can absolutely recommend BD's FACS Lyse reagents for nuclear staining, so if cost ain't a concern you might want to opt for that.
For the exact protocols check out some publications from my old lab.
Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...
Article Deubiquitinase MYSM1 Is Essential for Normal Fetal Liver Hem...
https://www.nature.com/articles/cdd2015140
All the best & good luck with your experiment,
Michael
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