Example 4
The measurement of mitochondrial membrane potential (MMP) with JC-9 was performed with two independent fluorescence channels (green and red fluorescence signals), as changes in MMP cause aggregation of the dye, with different fluorescence emission by the aggregated versus non-aggregated dye molecules. As shown in FIG. 6A, both red and green fluorescence initially increase in response to increasing valinomycin concentration, with a peak green fluorescence at˜0.05 μM valinomycin. At higher valinomycin concentrations, red fluorescence increases, with the red/green fluorescence ratio increasing. A unique aspect of this assay is that it correlates changes in MMP (JC-9) and cell death (SYTOXRED™) with the phases of the cell cycle (G1, S, and G2M).
An identical study to the one described before was conducted with exposing HL60 cells exposed to 1.23 μM idarubicin, an analog of daunorubicin which inserts into DNA and prevents it from unwinding during DNA replication and arrests cell division. As shown in FIG. 6B, the red fluorescence of JC-9 dye is significantly higher in cells treated with idarubicin than untreated control cells (first two data points on the left indicate control cells).
