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We are trying to sort G2/M cells of HT29s and the cells seem to be progressing
into G0 phase before we can complete the sort and return them to culture for
further analysis. We were wondering if anyone has tried this and might have
suggestions. We typically sort for about 4 hours and replace the collection
tube every hour with a tube from the ice and return the cells to the ice.
So far, the only thing we can think of is to keep the cells on ice constantly.
I think the sort is working because we can simultaneously sort G0 or S phase
cells cleanly. We check phase by fixing with EtOH and running with PI and an
internal standard. The sort is performed with Hoechst. Clumping may be a small
component, but it certainly does not adequately explain the results.
If anyone happens to know the amount of time that HT29 cells spend in G2, we
would love to know.
Thanks.
Kris
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