I am trying to compare the T cell subsets after ex vivo expansion at different time points. I want to stain the cells at the same time to reduce the variation between staining instead of just stain one or two sample everyday. Is it good to fix the cells right after cell collection and do the staining at the end or later after all samples were harvested? Does the fixation affect the recognition of antigen by the antibodies?
Dear Zhonghua,
The common procedure for surface markers is to stain the cells first (fresh), then fix them with PFA because the fixative makes surface epitops denature. In the other hand most of surface antibodies do not recognize fixed epitopes. Once fixed with PFA, cells can be stored for a few days at 4C, maximum 3 days is recommended.
But some people have a different opinion, they believe that it depends on the abundance of the surface marker. If it is abundant and you desire a qualitative answer, I mean positive or negative, you can fix cells before staining but it is not ideal. Personally I always work with fresh cells, stain and fix them with 1% PFA for maximum 24 hours.
Best,
Alaleh
Thank you for your suggestions, Alaleh. I also checked other posts and found similar suggestions as yours. It seem I have to stick with my current protocol and spend times on repeating work.
Best
Hi Zhonghua,
Although I agree that staining fresh is a always a good idea, you do not always have that luxury. Most times I fix my cells and stain them later. I usually do 11 colors and have found it to be consistent between experiments, both in MFI and in cell % and cell #. Like with anything though if your fixation isn't consistent between the different time points its pointless to stain them together, as it will change epitope access between groups, similar to what was suggested above. One suggestion is would be to treat your cells at different time points so you collect at the same time. As long as you have an untreated group at each time point for baseline it should be okay.
Hope this helps,
-Nate