RE: Jack's
Thomas Delohery (t-delohery@ski.mskcc.org)
Wed, 5 Apr 1995 19:10:47 +0530
Dude, I'm a fan. I also thought sort blots would "revolutionize the
field". I was able to get someone to try it once, only once.
The guy had so much hemolysis in his sample the blots were
visible from the hemoglobin. Not much use and he never tried
it again.
We did use your cocktail of RNAase inhibitors to suspend fixed
cells stained with Chromomycin to isolate G1,G2,early and late
S-phrase fractions. We had good Northerns from 1 million cells
per fraction. Sorting fixed myoblast cells was a nightmare -
they were torn apart and trying to show sort purity results was
very problematic. We went to your paper after we tried the
protocol Dennis Young cited (below). The 5XSSC caused all the
cells to clump into huge aggregates. We did sort into the
5XSSC but we couldn't suspend the cells in it.
Cheer up - maybe it was an idea (assay) too far ahead of it's time.
FURTHUR.
- From Dennis Young:
Paranoia of RNAse's will help ensure good results. DEPC-treated sheath fluid, as
well as vigilant sample-tubing cleaning are a must.
See Cytometry 11:869-874 (1990).
(They used fixed cells, 5XSSC instead of the standard PBS and washed
sample-tubing with 10% Hydrogen Peroxide.)
tom d.
QUESTION AUTHORITY..............
on every fundamental precept proposed as an explanation of any
aspect of reality; because reality is a perception and therefore
inherently subjective and gaussian in distribution. (possibly Poisson)
(I said that.)
--
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Manager, Flow Cytometry Core Facility | Phone: (212) 639-8729
Memorial Sloan-Kettering Cancer Center | Fax: (212) 794-4019
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