I have fixed bone marrow cells of mice in 1.5% PFA for 30mins and after washing stored them in 4°C resuspended in FACS fluid. It's already been 2 weeks now and flow cytometric analysis will not happen any soon. Will it be better if I stored them in -80°C now for a long term storage? The cells are not stained with any antibodies yet. What shall I do if I have to store them in -80? Shall I remove the FACS fluid and simply store the cell pellets directly in -80°C? I'm worrying as it's already 2 weeks in 4°C and the cells might start degrading!
*No, I will not recommend storing fixed cells at -80C., It will be all mess. It will be non-scientific
*You may cryopreserve Bone marrow cells before fixation following simple cryopreservation protocol of cells
That can stay at -80C upto a year (I have tested) or in Liquid Nitrogen for many many years.
Good luck
Hello Sayak Manna
Once fixed in PFA, cells may be stored for a few days (not more than 3-4 days at 4 degree C). The antibodies may either not recognize or sub optimally recognize the surface fixed epitopes if you delay the staining procedure. If you are looking for cytoplasmic markers, you need to fix the cells first, then permeabilize and stain later. You may fix them immediately, and permeabilize the cells and stain when you are ready to run your assay. Permeabilized cells are more prone to degradation, so permeabilization of cells must be done only on the day of the experiment. So, for flow cytometry it is always recommended that you work with fresh samples.
Best.