Hello Flowers: We have started working with JC1 and have noted some interesting observations. The setup: First I should say that we are working with adherent mouse lung cells. We are pretreating the cultures(in culture) with several compounds that "reduce" the mitocondrial membrane potential(MMP). These are: Valinomycin@10uM for 10 minutes, FCCP@ 2uM for 10 and 30 minutes and H2O2 @ 300uM for 2hrs. We are also using a positive inducer of MMP, Gramicidin@10ng/mL for 10 minutes. We are examining the preps thoroughly under fluorescent microscopy before flow cyto. I have gathered as many papers as possible on this subject, but compensation is still a concern of mine. I have a high fl2-fl1, but it seems to discriminate best here when comparing the fluorescent microscopy observations. The observations I have are: We found compensation to work such that fl2-fl1=50%, while fl1-fl2 is 4.7%. We are noting a strong increase in JC1 aggregate formation(red) with the FCCP at 30 minutes and almost no change at the 10 minute exposure when comparing to happy untreated cells. We expected green depolarized MMP in both. Why? The gramicidin increased the red JC1 Aggregate formation, but not significantly. Why? Valinomycin seems to work well, however, under epi-microscope there is red formation amoung the plasma membrane. This affects the flow data. What is this caused from? I have attached three jpeg and was hoping that someone could comment on these. As we have seen, there doesnt really seem to be an increase in red in fl2, at any compensation, but rather a decrease in green. We are able to discriminate at this point but wanted an experienced JC1 user to comment on this. The jpeg key is below. FCCP=red gramicidin=red+green H2O2= All green Scott Tighe Vermont Cancer Center Flow Cytometry Core Lab Burlington, VT
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