One possibility seems to be reaction of the succinimidyl ester of CFDA SE (also but inappropriately called CFSE) with cell-surface amines followed by spontaneous hydrolysis of the acetates to the green-fluorescent product on the cell's external surface. We use this technique with other fluorescent succinimidyl esters to distinguish live cells (which are dimly fluorescent because they are only labeled on their surface) from dead cells (which are labeled throughout their volume. The CFDA SE that penetrates the cell's membrane can both get conjugated to intracellular amines and intracellular esterases hydrolyze off the acetates to the green-fluorescent product. I am not certain that this is the cause but it is plausible. The fact that these do not divide, however, may indicate another cause. If your cells are still alive then the fluorescence of externally reacted CFDA SE (and labeled cells that died) can be almost totally quenched by trypan blue but the internally reacted CFDA SE should not be quenched, at least in a short time frame. Anti-fluorescein also quenches surface-reacted fluoresceins but not internal fluorescein in live cells. Presumably you should not see quenching with either reagent if the experiment is working well. I am not certain if others have observed this potential problem with CFDA SE. Jeffrey Dorfman wrote: > Dear All, > I have been having a problem staining PBMC with CFSE for cell division > tracking. > > Whenever I label PBMC, I seem to get a second population that is dimmer than > the rest of the cells. They do not seem to be cells that have divided, as I > see this even after an overnight incubation with no overt stimulation. > > The cells are not T cells and have a FSC/90LS profile matching that of > lymphocytes. Often, these dim cells are as much as 20% of undivided cells in > the lymphocyte FSC/90LS gate and they persist without apparent division for > as long as I have tested: one week. > > I have seen this with two batches of CFSE, with a range of concentrations > and labelling times of CFSE and with PBMC from a variety of donors. > > Is anyone familiar with what causes this/what these cells are and more > importantly, what conditions to use to prevent this? I have never seen such > a thing when labelling a large variety of mouse cell types with CFSE. > > Thank you for any help you can render. > > Jeff Dorfman > > ------ > Jeffrey Dorfman, Ph.D. > Kenya Medical Research Institute > Wellcome Trust Research Laboratories > Post Office Box 230 > Kilifi, Kenya > > jdorfman@kilifi.mimcom.net > jeffreydorfman@yahoo.com > > Phone +254 125 22063 > Fax +254 125 22390 > Mobile+254 733 86.23.10 > Home +254 125 22053 > > UK mobile +44 7814 574-997
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