How long can live cells labelled with fluorescent antibodies and fixed for subsequent analysis last before the fluorochromes degrade? Are there any methods to label cells for cell sorting/phenotyping and freeze them for later analysis months/years later?
I wouldn't trust fluorochromes to be stable if frozen for months/years. You can use 2% paraformaldehyde in your flow buffer in a final step when staining your cells as a fixative to crosslink your antibodies to the surface of your cell, and these can be kept at 4 degrees for up to a week. Maybe you can freeze the cells before staining them, and then thaw them when needed and stain at that moment.
Hi David,
I have stained, fixed cells and covered in aluminum foil then stored them at 4 degrees for weeks/months with no loss in fluorescence.
You can fix and then store/freeze cells to then stain and analyze later. However, it would not be a good idea to do this if you are interested in surface markers due to the possibility of the fixative denaturing the epitopes. For intracellular expression it is not a problem.
Good Luck!
Dear David,
I know that one of my collaborators have done that analysed for surface receptors after quite some time (several months), but I cannot remember if they were sained first and if they were frozen. Susanne Brix Pedersen at the Danish University of Technology. You might ask her about her experience.
I second John. I wouldn't trust cells stained and fixed for months together. I usually try to run flow with in 4 days after fixation. Just keep in mind that in order to do this, you need to completely cover your sample tubes with aluminium foil to prevent loss of fluoroscence. Also, if you are storing them in lidless tubes, cover them with parafilm to prevent evaporation of your sample.
Hi David, I represent STEMCELL Technologies here in Melbourne and we have a good tech tip that you may find useful on our website entitled "Fridge, Freeze or Fix: What to do if you can’t analyze your cells by flow cytometry immediately". See below, and ill be happy to discuss anything cell separation related as we have an extensive range of products in this area
http://www.stemcell.com/en/News/82a6b/March-Tech-Tip-Fridge-Freeze-or-Fix.aspx
interesting comments, thankyou all. More feedback is welcome.
regards
David.
Haven't tried it yet. It's still on the list of things we'd like to do if we weren't doing other things. Sorry.
Actually I should add, we have re-frozen purified immune cells in DMSO and FCS for cells we haven't been able to analyse at the time they were collected. This has worked beautifully for histone acetylation analysis. You can always default to this if you can't analyse your cells immediately.
In case anyone else is looking for it, the URL for the Igor's tech tip moved:
https://www.stemcell.com/fridge-freeze-or-fix.html