Calman Prussin wrote- >I am trying to identify eosinophils by flow cytometry taking advantage of >their avid binding of anionic dyes, such as FITC and Alexa. For one series >of experiments I only have the FL2 channel open on my Calibur to detect >the FITC/Alexa signal. I would like to try and use Alexa 532, which has an >absorption maximum at 532 and emission max at about 550. The FL2 set-up on >a Calibur consists of a 585/42 wide pass. > >Spectrum for Alexa 532 can be found >here: ><http://www.probes.com/servlets/spectra?fileid=11002p72>http://www.probes.c >om/servlets/spectra?fileid=11002p72 > >If bright staining is detected with an optimal flurochrome, what are the >chances I'll get anything usable with the Alexa 532? I guess I just need >to do the experiment, but was wondering if anyone had any thoughts or >experience. My experience is that eosinophils only stain with anionic dyes when they are fixed or permeabilized; the best way to identify them when they are not is by relatively high depolarized side scatter. However, there are two other fluorescence options. Eos autofluoresce more intensely than other granulocytes, and this fluorescence, equivalent to a few thousand molecules worth of fluorescein, should extend out to 585 nm. Also, eos express CD4, albeit relatively weakly. I can't tell you offhand which of the measurements just mentioned would be easiest to implement on a Calibur, but I'd be happy to discuss the subject. -Howard
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