Mitochondrial membrane potential quantification using TMRM (tetramethylrhodamine)

From a stock solution of 100 μM TMRM (Thermo, T668) prepared in DMSO a final concentration of 150 nM TMRM (diluted in water) was added to the top of the 35 mm NGM plates (3 mL NGM agar) already seeded with a bacterial OP50 lawn. TMRM solution was distributed over the entire plate surface and allowed to dry in a sterile hood with lid open for at least 45 minutes in order to ensure complete drying of the agar/bacterial surface. Since TMRM is light-sensitive, plates were covered with aluminum foil and were allowed to sit at 20°C for 24 hours before use.

Egg-lay synchronized populations of N2 and catp-6(ok3473) worms were grown at 20°C. Late L3 larval stage worms were picked and transferred onto TMRM plates for 24 hours at 20°C and then transferred to non-TMRM plates for 1 hour to remove excess stain. As a positive control we transferred 20–30 N2 worms from the 24 hours TMRM treated N2 plate onto FCCP supplemented non-TMRM plate for 1 hour. About 20–30 worms per strain were picked and mounted on agarose pad as mentioned above for visualization of TMRM fluorescence under Zeiss Imager Z1 fluorescence microscope using rhodamine filters. Quantification of images was performed using NIH ImageJ software. We quantified the fluorescence intensity of the TMRM stain in the whole body for an individual worm and normalized it to the body size of the worm. Graph represents the average fluorescence intensity ± SD intensity of all the worms shown in the image.