Measurement of Mitochondrial Activity

Mitochondrial membrane potential was quantified using the JC-10 probe. Cells seeded into 96-well plates were incubated in the absence or presence of H₂O₂ with or without Hb. At the end of the treatment, astrocytes were incubated in the presence of the JC-10 probe (10 µg/mL) at 37°C for 1 h, and then washed twice with PBS. In healthy astrocytes, the intact membrane potential allows the lipophilic dye JC-10 to enter into the mitochondria where it aggregates and produces an intense orange signal. In dead cells, mitochondrial membrane potential collapses so that the monomeric JC-10 probe remains cytosolic and emits a green signal. Fluorescence intensity was measured with fluorescence microplate reader and expressed as a ratio of the emission at 610 nm (orange) over 530 nm (green) to evaluate mitochondrial integrity (38). Experiments were carried out on 12 different wells from three independent experiments.