Are you sure you have correctly compensated the data? CFSE is quite bright and it is not uncommon for it to bleed substantially into the PE channel. If you use CFSE at a concentration of 5micromolar, which is often recommended by some protocols, you will see extremely bright (eg. later half of the 4th decade on a 'Calibur) CFSE fluorescence which is extremely difficult if not impossible to compensate out completely. For mouse cells, we use a concentration of 1micromolar, and some of my friends who stain human cells use even less. As a first step, I would recommend that you run some purified cells stained with CFSE alone and see if that mysterious "negative" peak appears in your PE channel. -jim James F. George, Ph.D. Division of Cardiothoracic Surgery Department of Surgery University of Alabama at Birmingham Birmingham, AL 35294-0007 Phone 205-934-4261 FAX 205-934-5261 jgeorge@uab.edu > > > ----- Original Message ----- > From: "Jake Kohlmeier" <jkohl@ku.edu> > To: "Cytometry Mailing List" <cytometry@flowcyt.cyto.purdue.edu> > Sent: Friday, November 09, 2001 7:39 PM > 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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