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I want to dual-stain cells of the osteosarcoma cell line SAOS-2 with
FITC/PI. The FITC-conjugated antibody is against a transfected surface
antigen for which I am achieving a transfection efficiency in the
neighborhood of 3-5% The only problem is that I keep losing a significant
percentage of the cells during the preparation protocol, presumably due to
damage/lysis. Normally, I harvest the cells (plated at 250K per 10cm plate)
with 0.1% EDTA in PBS for 10-15', spin @2K rpm for 5', incubate with 20 ul
FITC antibody in 80 ul media for 30', rinse in PBS + 1% FCS and spin @2K rpm
for 5', resuspend in 1.5 ml PBS, add 13.5 ml EtOH, fix @4'C for at least
1hr, spin @1.5K rpm for 5', and resuspend in PI stain (PI + RNAse + PBS + 1%
FCS).
Using this protocol, I usually lose about 60-70% of my cells between
harvesting to PI staining. I know that protocols such as this are commonly
used; I just haven't been able to figure out why I'm losing so many cells!
This loss is significant, because I need about 300,000 cells in order to
obtain enough cells positive for the surface antigen to allow DNA analysis.
The only thing I can think to do right now is use larger plates and more
cells for my
transfections, but I'm not still not comfortable with that high rate of cell
loss. Any suggestions or better protocols out there? Anybody trying similar
things with osteosarcoma cells?
Mark Clements
mclement@milbrandt.wustl.edu
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