RE: [Histonet] cytospins
I'm doing immunofluorescence on cytospins - and have had several
problems. First, we're looking at a rare cell from a FACS sort - about
cells per ml. We're getting a big loss when we cytocentrifuge - about
60% loss. Does anyone have a recommendation for a cytocentrifuge that
will conserve cells better than this?
Second, how important is it to fix cells immediately? Our FACS sorts are
done in Boston, then we travel about an hour to our lab. It is really
tough to fix cells in Boston - our procedure is Cytofix at 4C for 20
minutes, then acetone at 4C for 10 minutes, followed by PBS wash, block,
and overnight primaries. Will we experience serious cell deterioration
if we don't fix them for an hour to two hours after sorting?
I really appreciate any input. Melissa
I had similar problems when doing my Masters; cell loss from Cytospins
in poorly cellular specimens is a real problem. I was never sure if the
cells went into the filter paper or if they fell off. Urine preparation
has similar problems; they are poorly cellular and very watery. The
usual method is to add bovine albumin so when you spray fix the protein
glues the cells to the glass but it was still never satisfactory. We
tried Nuclepore preparations and they were better but you had the filter
to look through.
Liquid based cytology IMHO is the only way to do this; it solves the
fixation problem as they should be fixed ASAP and harvests many more
cells. If you can't do that then oddly you might get better results by
spinning the whole urine sample down and doing direct smears fixed ASAP.
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