Post Tue Oct 27, 2020 8:55 am

Multiple freeze thaw cycles on fixed cells

Hi All,

I'm working on making a positive reference anchor sample for batch correction and I will need to combine multiple tissue types in order to get a positive signal for all markers in the panel. I would like to be able to use cells from sources that I have previously phenotyped and are already frozen at -80C, but that would involve thawing fixed cells, mixing cells from different sources, and then aliquoting and refreezing at -80.

Our cells are fixed before freeze using stable-lyse2 stable-store2 (SLSS) from smarttubeinc. The plan is to thaw the SLSS cells at 4C as indicated by the vendor protocol and then refix in 1.6% PFA before mixing cell types and refreezing in aliquots. Aliquots will later be thawed one at a time (one per batch) and barcoded and stained with the experimental samples.

Does anyone have experience with refreezing fixed cells?

I intend to thaw a single tube of cells and refix and refreeze to test it before attempting to thaw and mix multiple samples. I'll run CyTOF on these twice fixed cells before attempting to thaw and mix cells for the anchor sample.

Any insight would be much appreciated!