Halo assay to assess mitophagy

Generating cells expressing mitochondrially targeted Halo-GFP using pSu9-Halo-mGFP from Mizushima lab (Addgene #184905; DOI: 10.7554/eLife.78923).

Seed HeLa cells the day before the treatment day in 6 well plates.

• Each well contained 2mL of growth media;
• Seed 350,000 cells for penta KO expressing BFP-Parkin and GFP-OPTN or -NDP52 and 380,000 cells for other knockout lines such as ATG13 KO/penta KO expressing GFP-NDP52;
• Adjust the number of cells of other cell lines, so that the next day they are all in similar confluency with penta KO expressing BFP-Parkin and GFP-OPTN or -NDP52.
  Note

  Remember to include a set of samples as your non-mitophagy-induced controls

The next day, make sure the seeded cells are spreading out (not concentrated in the middle of the well because this can affect the results).

Aspirate off the old media and treat each well with 1mL growth media containing 50nanomolar (nM) TMR-conjugated Halo ligand.

Incubate in a normal TC incubator for 0h 20m 0s.

Aspirate off the media and wash thoroughly.

Aspirate off the media and wash thoroughly with 1x PBS. (1/2)

Aspirate off the media and wash thoroughly with 1x PBS. (2/2)

Harvest the non-mitophagy-induced samples immediately by scraping (see step 8).

For mitophagy-induced samples, treat each well with 2mL of growth media containing 4micromolar (µM) Antimycin A, 10micromolar (µM) Oligomycin and 10micromolar (µM) QVD for desired period.

After the treatment, harvest the cells On ice by scraping.

Pre-chill eppies and 1x PBS On ice.

Aspirate the media thoroughly from the wells, wash the wells with 1mL of cold 1x PBS*, aspirate off the PBS and add 1mL of cold 1x PBS.

After that, use a plastic cell scraper to scrape all the cells off the wells (I use one scraper for each well. You can wash and reuse them again). Transfer the cells-containing PBS to eppies.

Centrifuge the eppies at 3000x g,4°C. Aspirate off PBS.

Quickly centrifuge for 0h 0m 10s to spin down the residual PBS. Aspirate off all the PBS.

Lyse the cell pellets in lysis buffer and boil the samples at 99°C with shaking for 0h 7m 0s .

Let the samples cool down and spin at max speed (Room temperature) for 0h 1m 0s.

Estimate the protein concentration by nanodrop.

Aliquot 25µg of each sample into a new eppie and add 1x LDS to make up to 15µL.

Set up the gel tank with MOPs buffer.

• The inside chamber should be filled with 1x MOPs supplemented with antioxidants.
• The outside chamber doesn’t need antioxidants.
• Wash each well with a glass syringe.

Load markers and samples into the wells and run at 100V for 0h 10m 0s and 190V for 0h 55m 0s.

Gels were then subjected to wet transfer onto PVDF membrane using cold NuPAGE transfer buffer containing 20% Methanol for 1h 0m 0s at Room temperature.

After transfer,

Incubate PVDF membrane with PVDF destain buffer on a shaker at Room temperature for 0h 2m 0s.

wash with PBS/Tween:

wash with PBS/Tween for 0h 5m 0s. (1/3)

wash with PBS/Tween for 0h 5m 0s. (2/3)

wash with PBS/Tween for 0h 5m 0s. (3/3)

Block with blocking buffer for 0h 15m 0s.

Remove blocking buffer,

Rinse:

Rinse with PBS/Tween. (1/2)

Rinse with PBS/Tween. (2/2)

Wash:

Wash with PBS/Tween for 0h 5m 0s. (1/2)

Wash with PBS/Tween for 0h 5m 0s. (2/2)

Wash with 1x PBS for 0h 5m 0s.

Cut the PVDF membrane and put appropriate parts into different antibodies (in this case, it’s VCP (1/1000) and HALO (1/1000) antibodies made up in 3% BSA in PBS/Tween) to incubate on a 4°C shaker 0h 5m 0s.

The next day, recycle the antibodies back to their tubes,

Wash:

• wash the blots with PBS/Tween for0h 5m 0s. (1/3)
• wash the blots with PBS/Tween for0h 5m 0s. (2/3)
• wash the blots with PBS/Tween for0h 5m 0s. (3/3)

Incubate with appropriate HRP-conjugated secondary antibodies (1/10000 for HALO and 1/5000 for VCP) made up in blocking buffer for 1h 0m 0s.

Wash the blots and develop.

Wash the blots,

wash the blots with PBS/Tween for 0h 5m 0s. (1/2)

wash the blots with PBS/Tween for 0h 5m 0s. (2/2)

Wash the blots with 1x PBS for 0h 5m 0s.

Develop the blots with ECL prime.