Dear Tim,
it is possible to do this experiment. Even a combination of CFSE and
intracellular staining. The concentration of CFSE is important. We use
standard 0.5 uM for the in vitro cultures. High(er) concentration will
influence the FL-2 to much and compensation in almost impossible. And we
combine this with PE, PerCP and APC with good results. You can find some
results at http://home.wanadoo.nl/flowcytometry/ under proliferation. The
sharp peaks can be due to several reasons, but as you can see on my site you
can also get them with 0.5 uM. The smaller (more homogeneous) the
(sub)population of cells, the sharper your peaks will get. It is difficult
to give you directions in voltage levels and compensation, you should manage
with a lower concentration CFSE.
I 'll be glad to help you with any question you might have. Kind regards,
Joost Schuitemaker
____________________________________________________
J.H.N.Schuitemaker
Laboratory manager / Senior Research Technician
Cellular Immunology Group
Cell Biology & Histology
Academic Medical Center
P.O.box 22700
1100 DE Amsterdam
The Netherlands
Telephone +31 (0)20 5664960
FAX +31 (0)20 6974156
Internet: http://home.wanadoo.nl/flowcytometry
http://www.amc.nl
-----Original Message-----
From: Tim Denning [mailto:tdenning@liai.org]
Sent: vrijdag 27 september 2002 19:22
To: cyto-inbox
Subject: CFSE 4-color
Dear Group,
I am not very familiar with CFSE analysis, much less with 3 additional
colors, so I was hoping someone might offer me some help. Thank you very
much for your time and help in advance.
Can anyone help me with instrument setting for 4-color with CFSE on the
FACSCaliber? I am interested in general voltages and compensation to make
sure I am in the right neighborhood (currently I think I am not even in the
right country so-to-speak). The person showing me how to do this is labeling
cells with >5uM CFSE (the more/greener the merrier method?) and during
analysis this person is greatly increasing the FL1 voltage and then
compensating 99.9% between FL1 to FL2 and quite high between other channels
as well. The person doing this tells me that using less CFSE is not good b/c
the peaks will not be "tight" and I will not be able to see all the
divisions clearly. Since I am a novice at this I understand his reasoning?
This person is incredibly bright and really knows his science, so I can't
imagine he doesn't have a reason for his method, maybe I just can't
understand it myself. I am attempting to simultaneously stain CFSE-labeled
cells with PE, PE-Cy5, and APC and I not having much luck. My experimental
set-up is labeling freshly isolated thymocytes with CFSE, expanding them
with various cytokines in vitro for 4 days during which time they clearly
undergo 3-5 divisions. I understand that my CFSE concentration (>5 uM) may
be WAY too high and I am working on trying various dilutions 2.5, 1, 0.5,
0.1 uM on my own. So here are my questions:
1. Is there a preferred dilution for in vitro studies and with that dilution
what are the general (I don't need exact numbers, just ballpark) voltages
and compensation values I might expect to use?
2. Is there a trick to get those beautiful, sharp peaks I sometimes see in
the literature?
Much thanks to the group and my apologies if my questions are repetative
and/or sophmoric.
Tim
Timothy L. Denning, Ph.D.
La Jolla Institute for Allergy and Immunology
Division of Developmental Immunology
10355 Science Center Drive
San Diego, Ca 92121
(858) 678-4534 (tel)
(858) 678-4595 (fax)
tdenning@liai.org
http://www.liai.org
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