JC-1 staining for mitochondrial potential (ΔΨm)

The assay was performed as previously described [59]. Briefly, assay of ΔΨm was performed using JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetra-ethyl benzimid-azolylcarbocyanine iodide, catalog no. T-3168, Molecular Probes) dissolved in DMSO at a concentration of one mg/ml. Age synchronized day one and day ten hermaphrodite and males were suspended separately in PBS containing freshly prepared 2% paraformaldehyde and incubated for 20 minutes for fixation, followed by washing with M9 buffer. Samples were subsequently transferred to micro centrifuge tubes containing 1µM JC-1 reagent and incubated at 37 °C in a 5% CO₂ incubator for 45 minutes. Samples were washed and suspended in S basal buffer, placed in 96 well black bottom fluorescence plates. Around 100 worms were used per well per replicate. Measurements of red fluorescence (excitation 550 nm, emission 600 nm) and green fluorescence (excitation 485 nm, emission 535 nm) were performed using a fluorescence plate reader (Tecan, infinite M200). The experiment was repeated thrice.