TITLE: Halo assay to assess mitophagy
AUTHORS: nguyen.tha

[DESCRIPTION]
This protocol describes how to assess mitophagy using Halo assay developed by Mizushima lab (DOI: 10.7554/eLife.78923).

[STEPS]
SECTION: Procedures
1. Generating cells expressing mitochondrially targeted Halo-GFP using pSu9-Halo-mGFP from Mizushima lab (Addgene #184905; DOI: 10.7554/eLife.78923).
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2. Seed HeLa cells the day before the treatment day in 6 well plates. 
Each well contained 2 mL 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. 
 

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3. 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).
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4. Aspirate off the old media and treat each well with 1 mL growth media containing 50 nanomolar (nM) TMR-conjugated Halo ligand. 
 

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5. Incubate in a normal TC incubator for 20 min.

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


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8. After the treatment, harvest the cells on ice by scraping. 
 


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9. Lyse the cell pellets in lysis buffer and boil the samples at 99 °C with shaking for 7 min .
 


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10. Let the samples cool down and spin at max speed (Room temperature) for 1 min.

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11. Estimate the protein concentration by nanodrop. 
 

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

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13. 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.
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14. Load markers and samples into the wells and run at 100V for 10 min and 190V for 55 min.

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15. Gels were then subjected to wet transfer onto PVDF membrane using cold NuPAGE transfer buffer containing 20% Methanol for 60 min at Room temperature.

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16. After transfer, 
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17. Remove blocking buffer, 
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18. 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 5 min. 
 


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19. The next day, recycle the antibodies back to their tubes, 
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20. Wash the blots and develop.
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8.1. Pre-chill eppies and 1x PBS on ice. 
 


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



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8.3. 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.
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8.4. Centrifuge the eppies at 3000 x g, 2 min, 4 °C. Aspirate off PBS.

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8.5. Quickly centrifuge for 10 s to spin down the residual PBS. Aspirate off all the PBS.

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6.1. Aspirate off the media and wash thoroughly with 1x PBS. (1/2)
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6.2. Aspirate off the media and wash thoroughly with 1x PBS. (2/2)
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6.3. Harvest the non-mitophagy-induced samples immediately by scraping (see step 8).
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16.1. Incubate PVDF membrane with PVDF destain buffer on a shaker at Room temperature for 2 min.

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16.2. wash with PBS/Tween:
wash with PBS/Tween for 5 min. (1/3)
wash with PBS/Tween for 5 min. (2/3)
wash with PBS/Tween for 5 min. (3/3)

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16.3. Block with blocking buffer for 15 min.

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17.1. Rinse:
Rinse with PBS/Tween. (1/2)
Rinse with PBS/Tween. (2/2)
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17.2. Wash: 
Wash with PBS/Tween for 5 min. (1/2)
Wash with PBS/Tween for 5 min. (2/2)

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17.3. Wash with 1x PBS for 5 min.

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19.1. Wash: 
wash the blots with PBS/Tween for5 min. (1/3)
wash the blots with PBS/Tween for5 min. (2/3)
wash the blots with PBS/Tween for5 min. (3/3)
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19.2. Incubate with appropriate HRP-conjugated secondary antibodies (1/10000 for HALO and 1/5000 for VCP) made up in blocking buffer for 60 min.

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20.1. Wash the blots, 
wash the blots with PBS/Tween for 5 min. (1/2)
wash the blots with PBS/Tween for 5 min. (2/2)

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20.2. Wash the blots with 1x PBS for 5 min. 

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20.3. Develop the blots with ECL prime.