Storage of cell samples before flow cytometry - (May/13/2006 )
I am preparing cell samples from live animal tissue for flow cytometry, which I don't have any experience with.
A problem is, the flow cytometry facility is at a diffent location from where I live so I can not run it immediately after I prepare the samples. In other words, after I get the samples ready to go, I need to store them (maybe for a few days). Do you have any suggestions on how to do this?
The experiment is a simple cell-sorting based on DNA content (DAPI-staining or something like that). I can prepare dissociated cells or possibly isolated nuclei. The specifc questions are:
(1) Can I freeze-store the samples before flow-cytometry?
(2) If yes, what kind of buffer should they be in? For other applications, I usually freeze nuclei in glycerol-containing buffer, but I am wondering if the presence of glycerol in the buffer is acceptable for flow cytometry.
(1/2): I haven't worked with DAPI, but I do not freeze any cells before cytometry. Freezing will most likely destroy your membranes... Just put them @ 4°C in an isotonic solution (PBS with/without FCS/BSA).
Thank you for the response vairus.
I'm doing a rather peculiar experiment here so let me clarify a little.
It doesn't matter if the cell membranes are destroyed or the cells are dead...as long as the nuclei are intact and I can sort them according to the DNA content.
Keeping the cells at 4c may be ok, but I worry that the cell cycle may possibly proceed at 4c. The purpose of this experiment is to obtain a "snapshot" of cell cycle profile at the moment of the sample preparation. Sample preparation here means either (1) extensive trypsinization (this is necessary to dissociate individual cells from the tissue) or (2) isolation of nuclei (which are also "sortable"). Either way, a storage step is unavoidable, which leads to my original question.
Any additional advices would be much appreciated.
I personally don't think cells will evolve a lot @ 4°C in PBS. Ice crystals formed by freezing might also destroy your nuclear membrane.
Maybe try fixation in 0,5% (or even less, I've gone down to 0,3% and all was fixed in approximately 10 minutes) paraformaldehyd in PBS for a couple of minutes, then wash extensively, dissolve in PBS and store @ 4°C.