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Tim O'Neill (tim_oneill@merck.com)
Help! PI Staining: Events/second problem
Hello all,
I have been doing standard FACScan PI analysis for DNA content
using cultured rat fibroblasts and a human carcinoma line.
My problem is that when I go to analyze, my sample settles
in the tube extremely easily and I constantly have to take off
the tube, resuspend, and keep collecting until I get 10-15,000
events, which takes forever! The last time I did this, I could
barely collect 2000 events. Is it that I just did not seed
a high enough concentration of cells?
Regardless of what my cell count is when I harvest, I resuspend
or concentrate to 2 million/ml.
My basic procedure is as follows: trypsinize, resuspend in media,
pellet at low speed (1200rpm), resuspend at 2 million/ml in PBS,
add equal volume of 70% ethanol overnight or longer, pellet cells
at low speed (1200rpm), resuspend in PBS, aliquot 1.0 ml of this
to a flow tube, spin low, resuspend back up in 1.0ml of PI staining
solution from Becton (50ug/ml), add RNAse (100ul of 0.5mg/ml),
incubate at 37C for 30 min, set on ice until ready to analyze.
I haven't done any vortexing. Should I be?
Or does this sound like an instrument problem to anyone?
Is there anything obvious here that I am missing or doing wrong?
I do get pretty good histograms once I collect.
Thanks in advance for anyone's help.
Tim
Merck Research Labs
(tim_oneill@merck.com)
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Posted by
J.Paul Robinson, Purdue University Cytometry Labs
Professor of Immunopharmacology
robinson@flowcyt.cyto.purdue.edu PH:765-494 6449 FAX:765-494 0517
web http://www.cyto.purdue.edu