Hi everyone.
I would like to isolate some PBMCs which I stained with CFSE and cultured for 72 hours, however I won't be able to analyse them with flow immediately, possible after 48hours, is it possible for me to wrap them in aluminium foil and store them at 4 degrees without fixing the cells for those 48 hours? I understand fixing with paraformaldehyde may reduce the brightness of the stain, that's why I am a bit reluctant in fixing them. We have previously stored some cells stained with CFSE in PBS overnight and analysed them with flow the next day and got some good data.
Thanks
Hi Vicky,
I don’t think it’s advisable to leave the stained PBMC's for such a long time after staining and before running them on flow. There is every possibility of cell death which will hamper your flow data though the cells are stained. U can fix the stained cells for 10min after staining and wash them twice, resusupend them in FACS buffer (or whatever your staining buffer is). Once the cells are fixed, there's no reason to leave them in fixative. Moreover leaving the cells in fixative for a long time will damage your epitopes can definitely mess up the fluorophores. Keep them up to 48 hours in 4C after fixation and the cells will be okay. Or you can keep the stained and fixed samples covered in foil, preferably in a dark.
Fixing is definitely not a good idea you can leave them at 4 degrees but mortality of cells could be an issue over longer periods of time. I am sure the cells will not divide at that temperature.
I agree that you'll probably have a considerable amount of cell death if you leave the cells at 4 degrees for too long a time. If you decide you have no other choice you might consider staining cells with a viability stain for Flow such as Ki67, so that you can isolate the live cells for your particular stain.
I agree with Vinod, however I will not recommend to fix them. You cannot compare fixed cells to non fixed cells, fixation should induce some changes.
I wouldn't leave PBMCs for so long without fixing as you will get a lot of cell death...
Thank you very much for your answers. I was considering staining the cells with 7AAD to determine the viability as well as gating them out when analysing the data. But it seems that it would be better to run flow immediately
Yes, running the flow immediately is definitely the best way to go. I am a bit afraid that, after such a long time at 4°C, gating out dead cells from your analysis would skew your results since you want to measure cell proliferation for which cell viability and senescence are very important parameters...
I agree with Vinood. If you need to fix them then do it for a short time and them give them a couple of washed and store in 4 degree overnight or 48 hours. This should not have any impact on your cells.
I would not recommend this plan. The father you get from the in-the-organism time point before fixation, the less reliable your data will be. It may have a shiny signal, but it won't be reflecting accurately the state of the cells.
Vicky... Running the stained cells on flow without fixing is the best option and that's what any one from any corner of the world will suggest you. But being specific to your question, you are after an alternative option with which the outcome of the experiment will never be 100% and can't be comparable to the std option. Since you want to measure cell proliferation, look at what Hassan, Damien, Maureen and me mentioned. Hope it make sense. Good luck ...
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