CD4/CD8 Staining Fails Post-Fixation in Some Donors – Has Anyone Seen This?
Hey everyone,
I am encountering a weird problem with my FACS panel for human PBMCs and hope for some insights.
Last week, I stained the panel and it worked well. However, when I stained today, neither in my single-stained cells nor in my fully stained sample was there any signal for CD4 and CD8. All other markers (CCR7, CD95, CD45, CD45RO, CD28, etc.) worked, and I used the same antibody vials from my rack as last week. However I used a different healthy control.
In my protocol, I am staining the cells with some extracellular markers that I wasn't able to get working nicely post-fixation (CD95, CD28, CCR7), stimulate the cells for 15 mins with cytokines, then immediately fix with BD Phosflow Lyse/Fix Buffer, followed by methanol permeabilization and intracellular staining (looking for several pSTATs) and some other extracellular markers.
I am aware of problems arising from staining fixed epitopes, as well as effects of methanol fixation on fluorophores, which is why I thought I had arrived at a good compromise and found a good panel.
Have you encountered the issue that staining of some epitopes post-fixation is also dependent on the donor? Or do you have any other idea why this might be? It wouldn't be a problem to change the clone, as we have some more options on hand, but then I also don't know whether this new clone would be reliable. I was hesitant to stain more markers than necessary before the cells are fixed as I am concerned, this could cause some phosphorylation events by activating agonist signaling.
The antibodies I used were Anti-CD4 BUV496 (Clone SK3) and Anti-CD8a PE-Dazzle594 (Clone Hit8a). On the Thermo Fisher website, "Antibody clone performance following fixation/permeabilization," the CD4 clone is listed as one that should work post-fixation. The CD8 clone is not listed there, but it has worked post-fixation in the past for me, which is why I selected it. I also did a single stain for CD4 PE today with the clone RPA-T4, and this worked well.
I also should mention that I am quite new to flow cytometry.
I appreciate your input!