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
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