Cheryl, The following webpage provided by Andrea Cossarizza may be helpful: http://flowcyt.cyto.purdue.edu/flowcyt/research/cytotech/amfc/data/page13.htm In a nut shell, it is difficult to distinguish JC-1 monomers from J-aggregates without using flourescence compensation. And, of course, it's nearly impossible to set the compensation without the proper control samples. Unfortunately, there is little help in the literature and the compensation is not as straight forward as for immunophenotyping. The webpage cited above gives this "helpful" hint: "compensations have to be set according to your feelings". So, it seems, you adjust your compensation settings until your control samples look like those presented in Figure 2. (If anybody has better instructions for setting the compensation, I'd like to see them, too.) Even "Current Protocols in Cytometry" is lacking in this regard (see Unit 9.14, contributed by Andrea Cossarizza and Stefano Salvioli): "typical green-orange electronic signal compensation near 4% and orange-green electronic signal compensation around 10%". This is a far cry from the structured and reproducible method of compensation provided by Mario Roederer at http://www.drmr.com/compensation/ I've only looked at JC-1-stained mitochondria in cells. Eric >Someone has come to me wanting to evaluate mitochondria using the dye JC-1. >The reference she provided me with is Experimental Cell Research, 222, 84-94 >(1996), by Anrea Cossarizza, Daniela Ceccarelli and Alberto Masini. >(Functional Heterogeneity of an Isolated Mitochondrial Population Revealed >by Cytofluorometric Analysis at the Single Organelle Level) > >What we would be looking for is a shift in the emitted fluorescence from 530 >to 590 upon membrane polarization. Now here's my problem..... >The authors have provided quite detailed information on the fluorescence >detector settings that were used (FACScan), and they indicate compensation >in both directions between FL1 and FL2. I'm afraid I just don't understand >why compensation would be used in this situation of metachromatic shift. Is >anyone familiar with this method? And while I'm at it, I have not had any >experience so far with mitochondria and any helpful hints in advance would >be greatly appreciated. I'm anticipating that I may have some difficulty >finding the little guys and recognizing them above noise. > >Regards, >Cheryl /\/\/\_ Eric Van Buren, aa9080@wayne.edu \ \ \ Karmanos Cancer Institute and Immunology & Microbiology \_^_/ Wayne State University, Detroit, Michigan, USA
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