Hey all, It probably isn't necessary to compensate the green-orange signals in these JC-1 samples, but I feel that it improves your ability you define limits between apoptotic and non-apoptotic cells. Compensation should be set with a sample that is unaffected (or minimally) - - and therefore [mostly] orange - - and one where depolarization is maximized (i.e., ONLY green). These serve as negative and positive apoptosis controls for the assay, and effectively define the range wherein you should find your experimental samples. So, since - - in a properly designed experiment - - we have these control samples, we might as well use them to set comp. It may come down to a preference, but I think the data looks better - - and may be easier to interpret - - if you do. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> Thomas M Delohery <t-delohery@mskmail.mskcc.org> 05/15/01 07:46PM >>> >Michael Ormerod said: >I have not run this assay but I do not understand why you are applying >compensation between FL1 and FL2. The fluorescence of JC-1 shifts from red >to green depending on the concentration of the dye. I would look at the >ratio between red and green, without compensation and look for a shift >wwhen the mitochondrial membrane potential collapsed. > >Can anyone else comment on this aspect of the assay? I may as well comment given the plethora of responses. Our experience has been limited so I can't cite a reference. We are more familiar with the metachromatic dye acridine orange (AO) that also shifts from green fluorescence at low concentrations to red fluorescence at high concentrations where dye "aggregates" occur. AO accumulates in acidic compartments within viable cells where it is protonated and trapped due to the positive charge. These acidic compartments give rise to bright red fluorescence and the cytoplasm fluoresces green. I suspect JC-1 behaves in a similar fashion given the reports of "hyperpolarized" mitochondria with bright orange (red?) fluorescence. We do not use compensation with JC-1 or AO. After all, what is compensation for? We monitor FL1 (525/20nm) and FL3 (>650nm) and as Michael suggests, we determine the red to green ratio. td Paglin S; Hollister T; Delohery T; Hackett N; McMahill M; Sphicas E; Domingo D; Yahalom J A Novel Response of Cancer Cells to Radiation Involves Autophagy and Formation of Acidic Vesicles Department of Radiation Oncology and Flow Cytometry Core Facility, Memorial Sloan-Kettering Cancer Center and Electron Microscopy Service, Rockefeller University, New York, NY Cancer Res 2001 January 15;61, 439-444 -- -- The information below is valid until June 1, 2001. I have accepted a position at Rockefeller University and will be there after June 1, 2001. ============================================================================== Thomas M. Delohery |Internet: t-delohery@ski.mskcc.org Supervisor, Flow Cytometry Core Facility | Office Phone: (212) 639-8729 Memorial Sloan-Kettering Cancer Center | Lab Phone: (212) 639-5162 1275 York Ave. Box 98 | Fax: (917) 432-2333 New York, NY 10021 | ==============================================================================
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