Fluorescence microscopy

Analysis of mitochondrial membrane potential using JC-1 dye was performed essentially as described (Kim et al., 2007 (link); Macchi et al., 2013 (link)) with slight modifications. Briefly, muscles, fat bodies and midguts of 25 day old male flies were dissected in Grace’s insect medium (Sigma, USA). Dissected tissues were incubated in 5 mM JC-1 (BioVision, San Francisco, CA) solution for 1 hour at room temperature followed by mounting in Grace’s medium and imaging using fluorescence microscopy (Zeiss, Germany) and AxioVisionLE4_3 software. Green (λem = 527 nm) and red (λem = 590 nm) fluorescence filters were used for microscopy.

Mitochondrial membrane potential (Δψ) was determined using a MitoPT TMRM assay kit according to manufacturer’s instructions (ImmunoChemistry Technologies, Bloomington, MN). Briefly, serum-deprived VSMCs were exposed to PIO (30 μM) for 1 or 3 hr, or vehicle control. Subsequently, VSMCs were stained with tetramethylrhodamine methyl ester (TMRM, 200 nM, 20 min at 37°C in the dark), washed with 1 ml wash buffer, and then visualized by fluorescence microscopy (Zeiss, Thornwood, NY). In intact cells, the cationic nature of TMRM allows it to accumulate within the inner membrane region of polarized mitochondria, resulting in a marked increase in TMRM-associated orange fluorescence. When mitochondrial membrane depolarizes, TMRM gets dispersed throughout the cytosol at a concentration that yields minimal fluorescence upon excitation. Thus, mitochondrial membrane potential was assessed by examination of TMRM-associated orange fluorescence (Excitation/Emission: 548 nm/573 nm). Carbonylcyanide m-chlorophenylhydrazone (CCCP, 50 μM, 1 hr) a generic mitochondrial membrane depolarizer, was used as a positive control.