I tried an experiment where I took an adherent cell culture (grown on 10 cm plates) out of an incubator, drove them to another facility (about 1.5 hours away), administered a treatment, trypsinized, and permeablized and fixed them with PFA and ethanol before driving them back and analyzing through flow cytometry. In all, they were outside of an incubator and sloshing around for about 5 hours (although I had a hot plate and kept them at 37 degrees when they were out of the car).
Upon staining them with PI and prior to passing them through flow, the cells all became clumped together and the flow yield was very low.
My thought is that the stress outside of the incubator may have killed a significant proportion of the cells, and that the DNA from the lysed cells is causing the remaining population to clump. The next things I am thinking of trying:
1. Cutting down on the time that cells are outside of the incubator before they are treated and fixed (to minimize the number of dying cells)
2. Considering using Hepes or another buffered media (as pH may be responsible for the stress that's killing the cells)
3. Using DNAse or Accumax to decrease the cell clumping after fixation.
Number 3 makes me a little nervous because the assay that I am running after fixation is DNA based, and I'm worried that adding DNAse to the buffer will just chew up the DNA in these permeabilized cells.
Does anyone have any suggestions about how to minimize cell death and clumping in this set up?