Hi David, this is a little late but, We recently did quite a few experiments for conditions of data acquisition with JC-1. The bottom line became, for a given cell type: Positive control = FCCP (titrated to a maximum of mitochondrial membrane depolarization) treated cells, with orange fluorescence minimized using compensation (lots of it). Negative control = normal cells (untreated) with orange fluorescence maximized (that is, finding conditions which allow the largest delta between positive and negative controls in the orange channel. The stuff is always green. For treatment with various other compounds under these conditions, it is prudent to check for fluorescence of the compounds themselves. For example, FCCP at higher concentrations contributes to the green fluorescence of the sample Here is a website which we found helpful: (right under our noses!) http://flowcyt.cyto.purdue.edu/flowcyt/research/cytotech/amfc/data/page13.ht m If you have log offset capability, this allows an even greater delta. We used this system as an audit of depolarization and not as a quantitative measure of delta psi. I would be happy to discuss by phone as well. Cheers, Sam Sam Witherspoon sw11527@glaxowellcome.com Dept. of Receptor Biochemistry Tel. 919-483-3078 Glaxo Wellcome R&D Page 919-857-7768 5 Moore Dr. Fax 919-483-0585 RTP, NC 27709 > -----Original Message----- > From: David_C_McFarland@sbphrd.com [SMTP:David_C_McFarland@sbphrd.com] > Sent: Thursday, June 29, 2000 2:43 PM > To: Cytometry Mailing List > Subject: JC-1 > > > 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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