When you freeze cells you put them through the worst hell they can experience and still live.  The problem is that most do not live.  With cancer cells this is not a problem because the cells that survived the vitrification process should recover after thawing and will grow to sufficient numbers.  Primary cells will not do this (usually)

Freezing a significant deviation in most experiments and should be avoided.  Some cells freeze better than others (usually related to size and organelles).  if you have different types of cells that you are sorting then how do you know one population is not hit harder than another population?  Also, DMSO will change your cells and at 10% can be considered mildly toxic (depends on cell types).  If you do not have a controlled rate programable freezer then you will have no way to control the freezing process (HUGE variability here).  Then there is the actual freezing media.  I would buy a commercially prepared media because it is well defined and consistently produced.  But with this said.  Freezing is way to much variability for my taste and should be reserved for cancer cell stocks.

Hi Shani, even more important to the freezing is the thawing. Because here the cells will die and break up. This will end in free DNA and that will cause aggregation. So You should consider using DNase during the thawing and sorting process. I hope you will find the ansere helpful.

Best wishes

Michael

Thawing should be done as quickly as possible.   In the protocol I used, we added ~10 volumes of an extracellular buffer that was warm.  Usually done in a 37 C water bath with continual gentle swirling.  Whether a cell dies is dependent on two things: 1) the ability of the cell to recover its ATP generating capacity and 2) membrane integrity.  During the thaw membranes become very leaky and the NA :K Pump has to restore the ionic gradient (easily 40+ % of the ATP will be used on this single pump).  This phenomena is clear when you look at size of cell verses post thaw viability.  Small cells recover better than a large cell.   Now if you have large cells that are recovering poorly (implying large loss in cells during thaw) DNAase may be useful.  It is also important to note that cells freeze best in relatively high concentrations (2-20 million per ml).   All of this variability from cryopreservation  would preclude any analysis by flow cytometery.  If you are absolutely sure your cells will survive and the marker you are looking at remains  following the thaw, then maybe sorting is possible but the cells would need at least 6 hours recovery time before sorting because they are in a fragile state (I would assume but would test before any experiments could commence)