We do a lot of Immunophenotyping and would like to use some samples which are positive for rare markers as controls for future experiments. Is it ok to freeze those samples after staining and fixation? Has anybody done that? Could you please share the protocol?
You can fix using ice cold methanol and store at -20 (it will not freeze) for at least a week. It might work for longer duration but I haven't tried. But staining has to be done just before FACS. You may try methanol+some test cells at -80 for longer storage as methanol freezing point is -96C. Note: by adding cells, the freezing point may be lowered.
Most of the fluorochromes are destroyed when frozen. I think it is best to find a cell line that expresses markers that you want to use as controls (or you can transfect them as you want).
I agree with the colleagues, fixed samples could be stored for some time (let say around 2 weeks, in certain cases till one month). Fixation by cold ethanol or methanol could preserve them at -20°C without freezing, thus protecting cell integrity. Obviously the stainings have to be performed just before FACS analysis, to have the best signal for acquisition.
It is generally possible to store fixed cells for flow analysis but it may be difficult to standardize conditions for all markers. You are probably best off doing them fresh
If you want to store it for some weeks or more then prior staining of the samples won't help. After staining you can keep the samples maximum for 12 hrs at 4 degree C. The acquisition has to be completed after overnight storage post-staining. Freezing the immunostained samples even 24 hrs will surely nullify all the signal causing error in data.
We routinely receive cells post-fixed after immunological staining in PFA, kept at 2-8°C for up-to 2 weeks, although we are very keen about stability of cell-surface staining since we do Antigen quantitation by QFCM (e.g. CellQuant Calibrator - BioCytex, or QIFIkit, DAKO). A useful trick: wash-out your 1% PFA after one night at 4°C (or alternatively 1-2h at RT). Keeping the cells in PFA will dramatically increase autofluorescence. But 2 weeks is a limit difficul to go over.
Alternatively, you lay want to try Transfix (Caltag Medsystems, UK) to store your cells before staining. Again, that may work for one-two weeks max.
Thanks a lot for all your suggestions. I think we will stick to the two week limit as suggested by most of you. Thanks Philippe for suggesting the wash-out protocol. Our cells do tend to be a bit more autofluorescent in general so it would be risky to leave them in PFA for longer.
Most of the fluorochormes phase out when stored for few days. So staining and storing the cells is not a good option since you are looking for rare markers in the immunophenotyping experiment.
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