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Anyway, on to my latest tirade:
I disagree with the notion that synchronous cells should be avoided in cell
cycle
assays due to potential artifacts. With that line of reasoning we should advise
people investigating cell cycle regulation not to use cell lines. The fact is,
much of our current knowledge of mammalian cell cycle regulation has come from
studies involving synchronous cells.
The experimental protocol I recommended to Denis Snider was to work out
the conditions for cells blocked at the G1/S border (aphidicolin). After the
drug is removed, one can follow a pulse of S-phase cells and determine when
a substantial portion of the cells are leaving S and entering G2. Then to
sample frequent time points during this period for his RNA assay.
This approach has several advantages. Each sample would be highly enriched
for G2 and provide large numbers of cells quickly. Cell cycle analysis
provides
the percent G1, S and G2/M for each sample. The kinetic data helps verify G2
as opposed to late S and M. You could do a short sort of the G2/M fraction and
score mitotics with a microscope for a more exact percentage. There is no loss
or degradation of the RNA from fixation and sorting.
There are lots of difficulties in attempting to sort a G2 population, especially
if you want to recover RNA. The biggest problem is they can not be resolved
from late S or mitotics by fluorescence intensity. At least I've never seen
definitive proof of any fluorescent DNA stain that can distinguish late S, G2
and M. The best one can hope for in a G2 sort is a semi-quantifiable
enrichment.
In reading Martin Poot's post from Jan. 20, '96, one might get the impression
that Molecular Probes' SYTOX GREEN is able to differentiate between
late S, G2 and M phase cells. Is this the claim being made by Molecular
Probes about SYTOX GREEN?
td
walkin' the walk
-- ============================================================================== Thomas Delohery | Internet: t-delohery@ski.mskcc.org Manager, Flow Cytometry Core Facility | Phone: (212) 639-8729 Memorial Sloan-Kettering Cancer Center | Fax: (212) 794-4019 1275 York Ave. Box 98 | New York, NY 10021 | ==============================================================================
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