Hoechst staining for FACS Vantage

From: Naoyuki Uchida (nuchida@bccancer.bc.ca)
Date: Tue Jan 08 2002 - 09:52:13 EST


Hi, I am Nao in Terry Fox Laboratory, BC Cancer Agency.

I have a question on SP (side population) cell sorting using FACS Vantage.
Small population of BM cells which have Hoechst33342-low phenotype are
called side population cells and are enriched in hematopoietic stem cells.
We used to work with FACStar Plus for this sorting and recently swiched to
FACS Vantage. But since then, we have not got proper profile. It looks to me
that bigger population which usually locates right next to SP cells (cells
in G0/G1) moves toward Hoechst-low position and overlaps with SP. We could
not figure out what was going on, and somehow the profile changed into
normal all of a sudden. So we know that our machine is capable of
visualizing SP cells, but we are still not confident in performing this
sorting properly for next experiment. If there are somebodies who can think
of what was the reason why it did not work in the first place and what
procedure to take when it happens, please let me know.

Thank you

Nao



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