Orange mitochondrial membrane potential assay kit

Mitochondrial membrane potentials of dAdo- or dGuo-exposed (160 μM; 24 h) U937-derived macrophages were analyzed by using the Orange Mitochondrial Membrane Potential Assay Kit (Abcam) according to the manufacturer’s instructions. Mitochondria of live (non-apoptotic) cells accumulate the MitoOrange Dye in this approach while the fluorescence intensity decreases following the collapse of the mitochondrial membrane potential in apoptotic cells. Stained cells were examined via flow cytometry and the FlowJo software (BD Life Sciences) according to standard laboratory protocols. For the detection of cytochrome c in deoxyribonucleoside-exposed cells, cell lysates were generated as described above and fractionated using the Cell Fractionation Kit (Abcam). Mitochondrial and cytosolic fractions were subjected to immunoblotting as described before by using the following rabbit primary antibodies: α-Cytochrome c (α-Cytochrome c, 11940, Cell Signaling) and α-GAPDH (α-GAPDH, ab181602, Abcam; loading control). Immuno-reactive signals were revealed with a secondary antibody conjugated to horseradish peroxidase as indicated before.

HeLa cells (1 × 10⁴) were seeded in 96-well plates [33 (link)]. The culture medium was removed the next day, and 100 µL of C-CM, N-CM, or H-CM were applied to the cells. After 12, 24, or 48 h of incubation, the cells were stained using an Orange Mitochondrial Membrane Potential Assay Kit (Abcam, Cambridge, UK). The fluorescence ratio (Ex/Em = 540/590 nm) indicating MMP was measured using a Mithras2 LB 943 Multimode Reader (Berthold Biotechnologies, Bad Wildbad, Germany) [33 (link)].