Hi Rick, I am not sure that I have any answers for you but I can say that I have seen similar things with DiOC6(3), CMXRos and LDS-751, Things, as you have observed, always seem fine with control cells - we use Jurkats or HL60 - but test cells can be more problematical especially when using adherent cell lines. As I understand it, DiOC6(3) isnt that fluorescent in aqueous solution but fluoresces brightly when within lipid bilayers. One thing that we have learnt to be careful about when using these dyes to assess mitochondrial membrane potential (We also use JC-1 and TMRE.) is dye concentration (and cell number therefore). I am not an expert (I was hoping your query would flush one out) but if you are seeing an increase in fluorescence after CCCP treatment could it be that the dye concentration is too high leading to quenching which is diminished when the membranes are depolarised? For our control cells where we see a nice shift (reduction) in fluorescence we use 20nM DiOC6(3). I have no explanation for the apparent increase in PI positivity, but quite often with CMXRos in adherent lines, I see the 'apoptotic' population heading diagonally from the LR quadrant to the UL. I think the bottom line is that these aren't 'off the shelf' cytometric techniques and we need to apply some thought to how the dyes work, what is happening in the cells and what other possible explanations there can be to anaomalous results. Its Friday afternoon and my head hurts. Derek Richard K. Meister wrote: >I just ran an experiment (flow cytometry, but would expect the same results >by confocal) staining T47D breast Ca cells with DiOC6(3) and PI to detect >apoptosis. We had Jurkat cells stimulated with camptothecin for >controls. Also, m-chlorophenylhydrazone (CCCP) was used to trigger loss >of mitochondrial membrane potential in some of the controls. The Jurkat >cell results were exactly as expected. >The results of the T47D cells, on the other hand, were very difficult to >interpret. For those unfamiliar with this assay, the unperturbed cells >fall into the lower right-hand quadrant when DiOC is plotted on the x-axis >and PI is plotted on the Y-axis. It should also be noted that the T47D >cells grow attached to the flask and were trypsinized ( and washed 2 times) >treatment with CCCP or staining with DiOC and PI. >The results for the T47D cells were as follows. I gated on the viable cell >population based on fwd vs. side scatter. > >1. Untreated cells stained with both DiOC and PI were a little more >fluorescent for both stains than were Jurkat cells, so I adjusted the lines >that define the quadrants accordingly. 93% of the untreated cells were in >the lower rh quadrant. > >2. Treatment with CCCP, which should have moved the cells as a whole >across the line into the lower left hand quadrant, instead resulted in a >second DiOC peak forming to the right of the original (-) peak, both still >located for the most part in the lower rh quadrant. > >3. An attempt to induce apoptosis by depriving the cells of serum for 2 >days resulted in the cells (DiOC/PI) being located in the lower rh quadrant >with a few cells leaking over into adjacent quadrants. But, for the most >part, the cells remained located in the lower rh quadrant, but divided >between 2 peaks. > >4. Starvation plus CCCP not only resulted in a minor shift half-way across >the vertical quadrant line to the left, but also a shift completlely across >the horizontal quadrant line into the PI (+) space. Remember that these >cells were gated on the live cell population by scatter. Changing the >scatter gate to include only the "dead" cells resulted in a DiOC/PI quad >plot that was virtually indistinguishable from that generated through the >"live cell" gate. This pattern was consistent for a duplicate set of samples. > >Do any of you have any experience with this assay and/or with this cell >line? Any explanation of the results? ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 London Research Institute, e_mail: derek.davies@cancer.org.uk Cancer Research UK mobile: 07790 604112 44 Linolns Inn Fields, London, UK. Web Page: http://sci.cancerresearchuk.org/axp/facs/davies/index.html In tenebris lux *************************************************************************
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