Hi Kevin, I have used the reference Bird et. al. "Immunity" vol. 9. 1998 with success at labeling primary T lymphocytes that are then grown in culture. I can't remember the exact concentration of CFSE but know that it's no more than 5uM, and this concentration is not too toxic to our cells. Check for cell clumping (death) when adding the dye, this can be reduced by lowering cell densities when labeling and/or lowering the CFSE concentration. You should synchronize your cell line before addition of CFSE to get a clear starting point. You might also find useful the protocol for B cell CFSE labeling contributed by Lyons and Doherty in "Current Protocols in Cytometry." I'm sure you know this already, but I think it's worth mentioning that fluorescein contributes to the fluorescence inside the cell but you add/incubate the cells with the non fluorescent 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester [5(6)-CFDA,SE according to Molecular Probes], and not CarboxyFluorescein Succinimidyl Ester as might be thought of with the nomenclature CFSE. I am embarrassed to admit that I was fooled by this on my first unsuccessful experiment using this method. Best of luck. Chris Chris Groves Millennium Pharmaceuticals Inc. 75 Sidney Street Cambridge MA 02139 617-679-7495 groves@mpi.com Kevin Waddick wrote: > > The following message was originally sent to Gang Chen in response > to his thanking people who responded to his message entitled "Ask for > help". He was asking for help using CFSE on lymphocytes. Although I > sent the message twice, originally on 1/28/99, I never got a response. > > So, I have decided to go to the source of his help. Namely, the > correspondents to Cytometry e-mail. I hope that those who helped him > can also help me. > I didn't mention in my original message that I found concentrations > of CFSE as low as 1 ug/ml to be apparently highly toxic to the cell > lines when used for 10 minutes in the original loading of the cells. > > Kevin G. Waddick, Ph.D. > Parker Hughes Institute > 2657 Patton Road > St. Paul, MN 55113 > (651) 628-9988 x 224 >
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