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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