We have been doing SP studies for more than a year. The staining seems to
be the biggest source of difficulty. We have found that both time and cell
concentration are important. This is often a trial and error on the part
of the investigator. We have used this procedure for many types of tissues
and can consistently get great profiles once the details are worked out.
for most of our studies, we use Hoechst at 3-5 ug/ml and incubate the cells
at about a million per ml. Times vary between species but 60-120 minutes
seems to be very adequate. When we first started this project I got some
UV excitable beads to assure myself the alignment was good before trying
the cells.
Tessa Kerre
<tessa.kerre@ To: Cytometry Mailing List
<cytometry@flowcyt.cyto.purdue.edu>
rug.ac.be> cc:
Subject: SP cells
01/17/2001
11:04 AM
Hi all,
Some of my colleagues in the lab are trying to set up a protocol for
studying SP (side population) cells on a FACSVantage, using Hoechst
labelling.
They have been trying for a few weeks now (using mouse bone marrow
cells, but ultimately they want to use human cells), but have problems
with setting the UV right, and getting the typical SP pattern. Of course
they do not know whether the problem lies with their labelling or with
the cytometer settings.
Are there people who have experience with this labelling, and can help
my colleagues with references and practical guidance.
Are there labs close to Belgium where they can come and learn at the
spot how to set the Vantage?
Thanks a lot!
Tessa Kerre
Ghent University Hospital - Immunology Department
Belgium
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