Mito-Keima assay to assess mitophagy

Seed the HeLa cells the day before the treatment day in 24 well plates.

Control cells include unstained cells (without any fluorescence proteins), cells that only express GFP-OPTN or GFP-NDP52 (to set up compensation so that GFP signal doesn’t bleach into mtKeima signal.

The next day, make sure the seeded cells are spreading out.

Aspirate off the old media and treat each well with 0.5mL of growth media containing 4micromolar (µM) Antimycin A, 10micromolar (µM) Oligomycin and 10micromolar (µM) QVD for indicated times.

Treat the longest time points first.

2 hours prior to harvesting, feed the untreated wells with 1mL of warm growth media.

Aftrer treatment, harvest the cells by trypsinisation.

Pre-chill eppies On ice.

Aspirate the media thoroughly from the wells.

Wash the wells with 0.5mL of 1x PBS.

Aspirate 1x PBS and add 150µL of trypsin and incubate at 37°C for 0h 2m 0s-0h 5m 0s.

Add 300µL of growth media to each well.

Mix well with a P1000 and transfer the cells to cold eppies.

Centrifuge the eppies at 1000x g,0h 0m 0s for 0h 2m 0s at 4°C.

Aspirate off the liquid.

Resuspend the cell pellets in 150µL of FACS buffer, transfer to pre-chilled FACS tubes and keep the samples On ice in the dark for analysis.