I need to analyze some tissue samples by FACS, but the analysis have to be performed after several months after the sample is taken and I have noted that when freezing cells with 10% DMSO in FBS they can loose some surface proteins, so I was wondering if I could get the samples, fix them and freeze them until the moment I use them, so then I could stain and acquire them.
Does somebody know if this is possible? Thank you so much in advance!
I don't recommend to freeze it particularly for facs sample as DMSO and H2O crystals will result in isotonic damage , better to try keeping them at 4°C maybe doing your staining just befor analyse, the result of the staining will probably be poor but if your antibody is strong it may work, have to try.
Hello, Jose.
If the tissue is a separated cell suspension (i.e., blood), wash the cells in appropriate buffer containing BSA (PBS+2mg BSA/ml) to make 4% PFA-fixed cell suspension. Be careful not to make sticky cell pellet. Use lower centrifugal force. Completely clean remaining PFA by repeating washes. In final suspension add 4% PFA to make 1% PFA in about 100 ul cell suspension. Store at 4C until use. Regularly replace 1% PFA. Then it will preserve surface proteins. However, if it is non-blood tissues, isolate cells first and then do the same.
Good luck.
Thank you so much Sang Ho Lee!, do you know how much time can I store the samples with that approach?
Hi, Jose.
If you regularly replace clean 1% PFA in PBS buffer (every two weeks maybe), it would be OK even several months. Since PFA tends to evaporate and inactivate in longer term. Replacement is necessary. However, more prolonged preservation, I suggest storing cells in prechilled methanol (MeOH; -20C) at -20C for over one year. Washing cells in buffer is important in transferring cells to prechilled MeOH for first fixation, change with new MeOH after the first fixation to remove any remaining previous buffer solution.
Regards.
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