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------- Forwarded Message Follows -------
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
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web