MitoTracker Deep Red Staining Procedure

Live cells were incubated with 100 nM of MitoTracker Deep Red (Life Technologies, Grand Island, NY, USA) for 20 min under regular culture condition or left unstained as a negative control. Stained cells were washed with PBS, adhered to 10-well slides, fixed, and permeabilized as previously described [40 (link)]. Cells were blocked with Image-iT FX signal enhancer (Life Technologies) for 15 min at room temperature, and then either singly or doubly stained with primary antibodies. Subsequent labeling with Alexa Fluor-conjugated secondary antibodies and DAPI counterstain (Life Technologies) were performed to visualize primary antibodies and nuclei, respectively. Stained cells were viewed using the Olympus FV10i fluorescence confocal microscope. Images were analyzed using the Fluoview software (Olympus, Melville, NY, USA).