Jake, CFSE is extremely bright compared to other labels. As such, it crosses over very badly into the other channels, and requires lots of compensation. That "negative" you see (in the absence of CFSE) is most likely due to over-compensation. Three points . . . 1) Dye concentration: we've seen very effective staining at the low end of recommended concentrations - - 1.5 ug/ml. At this lower concentration, cross-over is more manageable. I suggest you titrate to the lowest concentration that works with your cells. 2) Compensation: most comp circuits are not linear . . . extreme signals (low and high) may be effected very differently at the same compensation setting. So, a comp setting that works well for a low signal may be inadequate for very bright signals. With CFSE, comp is usually fairly high. As the signal diminishes over time, the intensity may drop . . . when this happens, (even though the signal drop may be relatively small) comp level may actually be too high, possibly dragging some "positive" events into the negative area. The best you can do is to run control samples that will help you set the appropriate comp level for the brightest signal on that day. Alternatively, comp can be done in software (where it is actually linear), if that's available to you. Software comp, however, doesn't help if you want to sort . . . (editorial comment). 3) Filter: since this assay is so sensitive to signal cross-over and comp level, we've shifted our PE filter from the usual 575/30 bandpass to a 574/14 bandpass. This diminishes the PE signal, yes, but it also limits the CFSE cross-over to a far more manageable level. Most analyzers won't have the option of changing filters like this, but it's something to keep in mind for users that have open benches. There are other things that may improve the assay . . . using a SA-PE versus a PE direct to increase signal separation, using APC (no CFSE cross-over) . . . in the context of a multi (more than two) marker assay, these options are limiting on most instruments, so your best approach comes back to better management of the CFSE signal. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> "Jake Kohlmeier" <jkohl@ku.edu> 11/09/01 08:39PM >>> 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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