You may want to try an excellent dye for determining the inner mitochondrial potential disruption. TMRE sold by Molecular Probes ( no endorsement) is very bright and no problem of compensation. Simply incubate (1 million cells/ml) in complete tissue culture media along with 40 nM of TMRE for 20 min at 37oC. Wash once in staining buffer (Dulbecco's PBS + 0.2% BSA, filter sterilized; azide is not required if cells are analyzed within hours) and resuspend in the same buffer and keep the cells on ice till you run on a flow cytometer. I generally can get away with 100,000 cells per condition. I have used a variety of human cell lines as well as primary cells. There are a number of reports using TMRE out there. You may also want to check the website of Molecular Probes for full details and references. Good luck. Jay Sundararajan Jayaraman, Ph.D. Research Assistant Professor of Surgery Diabetes Research Institute-R134 Miami University School of Medicine 1450 NW 10th Avenue Miami, FL 33136 Telephone: 305-243-3700 Fax: 305-243-4404 > ---------- > From: Oughton, Julie > Sent: Thursday, May 9, 2002 3:21 PM > To: Cytometry Mailing List > Subject: JC-1 > > > > I just had a head-banging experience with JC-1 fluorescence. One of my > users > wants to examine the mitochondrial membrane potential of colon epithelial > cells, using JC-1 [final concentration = 2.5 ug/ml]. > > We are using a Coulter XL flow cytometer; collecting data in FL1 [530 > bandpass] vs FL-2 [575 BP]. We also collected data in FL1 vs FL-3 [620 > BP]. > Volts for each PMT was ~580. > > Negative control: Untreated cells > Positive control: Cells treated with 0.1mM valinomycin in DMSO > for 2 hours > > I am finding it very difficult to compensate JC-1 fluorescence. According > to > archived messages on this list, compensation can be quite tricky. From > what > I understand, the positive control should be producing green fluorescence > while untreated cells mostly orange. According to the fluorescence pattern > in figure 2 at: > > http://flowcyt.cyto.purdue.edu/flowcyt/research/cytotech/amfc/data/page13. > ht > m > why aren't untreated cells considered to be both orange and green? > > Unfortunately, the pattern of our JC-1 fluorescence (monomers vs > aggregates) > looks quite bizarre [see attached histogram, no compensation]. In > addition, > valinomycin treatment resulted in a large population of cells that > exhibited > low FS [see attached figure; these cells are color evented as red], > probably > apoptotic cells. And their fluorescence pattern was not what we expected. > > I would love to discuss our data with someone who has experience with > JC-1. > Any suggestions to help set proper compensation? Any comments? > > Thanks in advance! > Julie Oughton > Oregon State University > > > > >
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