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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