Mitopy1

Fluorescence image analysis was used to determine the relative levels of ROS. Cells were harvested and suspended at 1×10⁶ cells/ml in PBS. The relative levels of intracellular ROS were analyzed using the cell-permeable superoxide-sensitive fluorochrome 2′,7′-dichlorofluorescein (Invitrogen). Cells were incubated with CM-H₂DCFDA (2μM) for 15 minutes at 37 °C before analysis using a BD FACSCanto™ II flow cytometry (BD Biosciences). Mitochondrial ROS level was determined by using a specific mitochondrial H₂O₂ probe, MitoPY1 (Sigma). Cells were incubated with 10μM MitoPY1 and 100nM MitoTracker(a mitochondrion-selective probe, Invitrogen) for 30 min at 37 °C, then washed and analyzed by TCS SP8 confocal laser microscope (Leica). The fluorescent intensity of MitoPY1 was quantified using the NIH ImageJ.

The ROS and the mitochondrial ROS were measured using a previously standardized protocol.^{46 (link)} Briefly, 1 × 10⁶ cells were incubated either with 5 μM of cell-permeant, fluorogenic ROS sensor CellROX Deep Red reagent or with 10 μM of MitoPY1 (Invitrogen) in the culture media for 30 min at 37°C and subsequently the indicated treatments were initiated. Fluorescence was measured either at an excitation wavelength of 640 nm and an emission wavelength of 665 nm or at an excitation wavelength of 514 nm and an emission wavelength of 530 nm using the Fluostar Omega spectrofluorometer (BMG Technologies, Offenburg, Germany).