From: Claude Cantin (cantinc@oci.utoronto.ca)
Date: Mon Sep 28 1998 - 16:23:52 EST
A few weeks ago a few people posted concerns about post sort mortality and non- proliferating cells. It was suggested the sheath fluid might be the source of this problem. I was told that the sheath fluid marketed by Becton Dickinson in the US was toxic, but that marketed in Canada was non-toxic. My colleagues indicated no problems with the FACSFlow solution available in Canada. As it turns out, I now have a very serious viability problem with both primary and secondary cells sorted since our FACStar plus was upgraded with the Turbo Sort Option. Cell lines have suffered since installation of the TSO; now primaries also will not proliferate under conditions in which they used to do well. Mortality includes negatively sorted cells. Is anyone aware of a recent change in formulation of the FACSFlow solution? Is there any word of a bad lot on the market? Has anyone else experienced mortality problems since installation of a TSO? Anyone have any ideas? Post sort analysis routinely shows better than 95% recovered cells in the 'live' sort gate: the cells have the same FSC/SSC profile as non sorted cells (with little or no cellular debris), yet 24 - 48 hours later, these populations are non-responding or dead. Unsorted samples from the same animals survived well in the same culture medium as that in which the sorted samples were re-cultured, so whatever evidence there is seems to point to sorter related difficulties. Last week, primaries which in the past showed no difficulties surviving high speed sorting died; cell lines which suffered at higher pressures (30) PSI were sorted at normal non-TSO pressures (11 PSI) and survived quite well. Yet the same populations survived high speed sorting (same conditions: 30 PSI, 61K DDF, 70 uM nozzle, FACSFlow sheath) on 2 neighboring instruments. It's getting impossible to sort out coincidence from relevant fact! Standard TSO sorting conditions: 30 PSI sheath pressure; 70 micron nozzle; ~61,000 DDF; lowest possible deflection voltage; drop delay ~28 - 29; 6 - 8,000 events per second; large diameter (recently changed silicone) sample injection tubing. The instrument fluidics system hasn't seen bleach or ethanol in weeks: since this mortality started occurring I dared not use any bleach, not wanting to risk carry over, or potential toxins in our d2H2O supply. Only the sample injection tubing is flushed with 3% U.S.P. hydrogen peroxide, and rinsed with sheath fluid or collection medium (PBS+FCS). No reports of bacterial or mycoplasma contamination, though the latter has not been tested for; the 0.2 micron sheath fluid filter was recently changed (2 -3 weeks ago), and about 20 liters FACSFlow is used in this instrument per week. Desperately Seeking Solutions.... Claude
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