Dual fluorescent staining was performed to detect mitochondrial heat shock protein 60 (mtHSP60; Santa Cruz Biotechnology) and nuclei, using 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR, USA). Cells were cultured on IBIDI Micro-Slides (Applied BioPhysics, Troy, NY, USA), fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100, blocked with bovine serum albumin (BSA) for 1 hour, and immunostained with an anti-mtHSP60 antibody overnight at 4°C. The cells were then incubated with fluorescein isothiocyanate (FITC)-conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA), stained with DAPI for 3 minutes, and mounted with Fluoromount (Sigma-Aldrich Corp.). Cells were examined using a confocal fluorescence microscope (model TCS-SP5; Leica Microsystems CMS GmbH, Mannheim, Germany) with three lasers (argon, 488 nm; diode, 405 nm; Leica Microsystems). Mitochondrial morphology was determined in 100 individual cells and scored based on the filamentous and fragmented forms in each of five different experiments.