Good day to all. We've been trying to do some mitochondrial membrane potential experiments with cardiomyocytes. JC-1 was chosen based on information in: Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes. Anthony Mathur, et al. Cardiovascular Research 46 (2000) 126-138. We are using carbonyl cyanide m-chlorophenylhydrazone (mClCCP) as a positive control but running into difficulties. This paper states that "owing to the dual wavelength emission of JC-1, electronic compensation was used in this case to correct for spillage of the green (monomer) and orange/red (aggregate) fluorescence signals into the FL2 and FL1 channels, respectively". Can someone tell me how this was accomplished. After treatment with mClCCP, orange fluorescence in significantly reduced. However, this doesn't really give a single-color green control. So, how do you do compensation with this dye? I'm under the impression that compensation is critical if you are using change in MFI as your indicator. Or do you use standard particles that are about the same fluorescence intensity? As I said, mClCCP greatly reduces the mean orange fluorescence, yet doesn't seem to affect green fluorescence very much. One would infer that these would correlate and move in oposite directions. In addition, another treatment expected to decrease orange fluorescence actually increased above basal levels. I would be most appreciative of any info on this system that I could obtain. As always, many thanks in advance. David McFarland SmithKline Beecham Pharmaceuticals
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