1) Generation of cell lines stably expressing YFP-Parkin and mt-Keima.

1-1) Day1. Seed HEK293T cells in a 6-well plate.

1-2) Day2. When the cells reach 70-90% confluency, perform transfection.

Add 0.5 ml Opti-MEM into a sterile 1.5 ml microcentrifuge tube. Add 0.5 ug VSV-G, 1 ug Gag-Pol, 1.5 ug mt-Keima retrovirus plasmid or YFP-Parkin retrovirus plasmid. Add 9 ul of Lipofectamin LTX. Vortex for 30 sec. Let them sit for 30 min at room temperature. Replace the media in the 6-well plate with 1.5 ml fresh DMEM complete media. Add the transfection complex dropwise to the cells.

1-3) Day3. Change the media after 24hrs of transfection with 1 ml fresh DMEM complete media.

1-4) Day3. Seed target cells in a 6-well plate.

1-5) Day4. Infect the virus to the host cells.

Collect and put the virus supernatants into 1.5 ml sterile microcentrifuge tube. Quickly spin for 1 min at 1000 xg to remove cells or debris. Filtrate the virus using a PVDF Millex-HV filter and a 1 ml syringe. Remove the media from the target cells. Add filtrated virus to the target cells with 8 ug/ml polybrene and culture media up to 2 ml.

1-6) Day5. Change the media with 2ml fresh DMEM complete media.

1-7) Day6 (Optional). Check mito-Keima and YFP-Parkin signals under a fluorescent microscope.

2) FACS analysis of mitophagy

2-1) Day1. Seed the cells stably expressing mt-Keima and YFP-Parkin in a 6-well plate.

2-2) Day2. When the cells reach 70-90% confluency, treat the cells with 10 uM oligomycin, 4 uM antimycin A and 5 uM Q-VD for 6-24 hours at 37°C.

2-3) Day2 or 3. Wash the cells with 1ml PBS. Add 0.2 ml 0.05% trypsin-EDTA, and incubate at 37°C until the cells are taken off the well. Add 0.7 ml DMEM media to the well, mix and transfer the cells into a 1.5 ml microcentrifuge tube. Centrifuge for 1 min at 800 xg. Discard the supernatant and resuspend the cells with 1ml PBS containing 2.5% FBS. Transfer the cells into 5 ml round-bottom tube with cell-strainer cap.

2-4) Analyze mitophagy by FACS.

We use MoFlo Astrios cell sorter (Beckman Coulter) or BD LSRFortessa X-20 cell sorter (BD Biosciences). Sort YFP and Keima (pH7) double-positive cells.

Y-axis: Keima pH7 with Ex. 405_Em. 610-620. X-axis: YFP with Ex. 488_Em. 513-526. Perform ratiometric analysis. Y-axis: Keima pH4 with Ex. 561_Em. 610-620. X-axis: Keima pH7 with Ex. 405_Em. 610-620.

For each samples, more than 10,000 YFP/Keima double-positive cells are collected.