CFSE staining and unlabeled cells
rozenkov at netscape.net
Sun Feb 19 00:12:23 EST 2006
It would be of help to people trying to help you if you could post example pictures of stimulated and control cultures. As has already been correctly suggested, a picture of stained cells before culturing would also help. Otherwise, unlikely anybody will be able to finally solve you problem with this amount of information.
Generally, if you do not add any proteins to PBS (as you describe) 10uM CFSE for 10mln/ml MNC is a lot and should not require increasing CFSE concentration or decreasing cell density.
Anyway, it is necessary to see where your divided cell peaks are, where the undivided cell peak is, whether you can see separate division peaks and how many, why you have events in the first log, why they overlap with the sixth division, etc.
Do you use MNC and how much of RBC contamination do you have? I have once seen odd peaks due to RBC.
Vladislav Rozenkov, MD, PhD
From: enrico lugli <elugli at hotmail.com>
Sent: Wed, 15 Feb 2006 10:02:31 +0000
Subject: CFSE staining and unlabeled cells
Hi flow'ers community,
I performed CFSE staining of peripheral blood lymphocytes. I followed the protocols found in the literature, that is to say suspension of cells in PBS at a concentration of 10^7 cells/mL, staning with 10uM CFSE, incubation for 10 minutes at 37°C, washing with complete medium and plating. I stimulated cells with cytokines such as IL-7 and IL-15. At day 6, quite surpring, I found a consistent proportion of cells on the first decade of fluorescence adding to proliferating cells that diluted CFSE; I'm not sure they were proliferating cells at high rate, since they were found also in the unstimulated control. Have you got any suggestions? Have I to increase the concentration of CFSE or diminish the cell density at the time of staining?
Thanks to all,
Flow Cytometry Unit
Dept. of Biomedical Sciences
University of Modena and Reggio Emilia
41100, Modena, Italy
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