Jeff, First . . .have you tried this at 488nm? PKH26 is fairly bright stain . . . we usually see about a 3 decade shift from background. So, I wouldnn't worry too much about maximizing excitation. Remember, it's not so much signal intensity, but rather signal-to-noise ratio that improves resolution. Second . . . you could narrow the band range on your filter. When I need a better separation against a ~575nm signal, I'll use a 574/14 filter . . . helps when I use CFSE vs. PE by eliminating much of the green crossover from the extremely bright CFSE. Third . . . have you tried CFSE? Very bright, and I have a sense that it may be less toxic than the PKH dyes. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> Jeff Louie <jlouie@hsc.usc.edu> 03/15/01 02:42PM >>> I have a researcher who would like to utilize PKH26 as a tracking dye for rabbit rbc's survival studies. I have gotten reasonable separation of positives and negatives using a "PE" filter setup (555DLP beam splitter and 580/30 band pass) with 488 excitation on a MoFlo. The researcher is worried about cell toxicity of the PKH26 and wants to use as little as possible in his staining procedure. In an attempt to maximize fluorescence intensity, I've tried to "tune" the dye head laser (INNOVA 70) on the MoFlo to maximum excitation for PKH26 (551 nm) but seem to overwhelm the detection PMT with its 580/30 band pass. Any suggestions or advice on filter setup or use of PKH26 in this scenario is much appreciated.
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