Hi Jake, Possible answers to your questions include: a) you've co-purified another cell type in your CD4 positive selection (monocytes or something sticky) that do not respond to your T-cell-specific stimulation, and so don't divide (whereas those that do divide are CD3 positive T-cells). To check this against your hypothesis (CFSE interference) you should stain for CD3 and perhaps a panel of other leucocyte markers before you load with CFSE and see if anything changes when you load with CFSE. What's the FSC/SSC like for the non-dividing CD3 negatives? - you should be able to gate them out if they are monocytes. b) Probably "illegal" - presumably you've set your compensation so that CFSE alone goes straight along the FL1 axis and PE alone goes straight along the FL2 axis for a given pair of FL1/FL2 PMT voltages. If you then alter the voltages, the compensation required changes - increase FL2 PMT and the CFSE single positives will move upwards (the doubles will as well, but nowhere near as much) Ray > From: "Jake Kohlmeier" <jkohl@ku.edu> > Date: Fri, 09 Nov 2001 17:39:38 -0800 > To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > Subject: CFSE > > > Dear flowers, > Being a rookie in this field, I was wondering if anyone out there has > answers to some of the problems I have encountered with CFSE staining. When > I label purified human CD4+ T cells with CFSE and also stain with a second > marker (CD3 for example) I see a population of cells that have not divided > that appear negative for CD3, as well as some that are positive. In cells > that have undergone 1 or more divisions, there is no longer a CD3 negative > population. As I expect, when I run CD3 alone as a control all the cells > are positive. > > If I increase the voltage in FL2 when looking at CFSE and CD3 > simultaneously, I can "move" the CD3 negative cells so they are now positive > without affecting the intensity of the cells that were positive to begin > with at the lower voltage. So my questions are: > > Why am I seeing a CD3 negative population and why do I see it only in > non-dividing cells? Does this have anything to do with the fact that the > stronger CFSE signal from non-dividing cells is somehow interfering? > > Also, is increasing the voltage of FL2 a "legal" thing to do to make this > small population of cells stain positive for CD3? I want to be sure that by > doing this I'm not changing reality and the data are still of good quality. > Any suggestions are welcome. Thanks. > > Jake Kohlmeier > Department of Molecular Biosciences > University of Kansas > 1200 Sunnyside Ave. > Lawrence, KS 66045 > (785) 864-4029 >
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