May 24, 2023
Halo assay to assess mitophagy
- 1Laboratory of Michael Lazarou, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
Protocol Citation: nguyen.tha 2023. Halo assay to assess mitophagy. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpjzm8gzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 05, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 73537
Keywords: Halo assay, mitophagy, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000350
Abstract
This protocol describes how to assess mitophagy using Halo assay developed by Mizushima lab (DOI: 10.7554/eLife.78923).
Attachments
585-1231.docx
20KB
Materials
Buffers and reagents:
Growth media:
| A | B | |
| DMEM with 10% FBS | ||
| Glucose (Sigma, G8769) | 4.5 g/l | |
| 1x GlutaMAXTM (ThermoFisher, 35050061) | ||
| 1x MEM NEAA (ThermoFisher, 11140-050) | ||
| HEPES (1688449) | 25 mM |
- Antimycin-A (Sigma, A8674; made up in 100% Ethanol to 20 mg/ml),
- Oligomycin (Calbiochem, 495455; made up in DMSO to 10 mg/ml)
- qVD (MedChemExpress, HY-12305; made up in DMSO to 10 mM)
- TMR-conjugated Halo ligand (Promega, G8251)
- Lysis buffer: (diluted from 4x LDS (NP007; ThermoFisher)
| A | |
| 1x LDS | |
| 0.1 M DTT |
can be aliquoted and stored at -20 or -80°C.
- 4-12% Bis-Tris NuPAGE gels (ThermoFisher)
- NuPAGETM Antioxidant (NP0005, ThermoFisher; use 0.5 ml/ 200 ml of gel running buffer)
- 20x NuPAGETM MOPS SDS running buffer (NP001, ThermoFisher)
- 20x NuPAGE transfer buffer (NP00061, ThermoFisher)
PVDF destain:
| A | |
| 40% Methanol | |
| 7% Acetic Acid |
- 1x PBS
- 1x PBS/0.1% Tween20 (PBS/Tween)
- Blocking buffer: 5% skim milk in PBS/Tween (make fresh)
- VCP (Cell Signaling, 2649), HALO (Promega, G9211)
45% D-( )-GlucoseSigmaCatalog #G8769
GlutaMAX™ SupplementThermo FisherCatalog #35050061
MEM Non-Essential Amino Acids Solution (100X)Thermo Fisher ScientificCatalog #11140050
HEPES Buffer 1M Solution Cell Culture Grade MP BiomedicalsFisher ScientificCatalog #ICN1688449
Antimycin A from Streptomyces sp.Sigma – AldrichCatalog #A8674
OligomycinMillipore SigmaCatalog #495455
Q-VD-OPhMedChemExpressCatalog #HY-12305
HaloTag(R) TMR Ligand, 30ulPromegaCatalog #G8251
NuPAGE™ LDS Sample BufferThermo Fisher ScientificCatalog #NP0007
NuPAGE Antioxidant Thermo Fisher ScientificCatalog #NP0005
NuPAGE™ MOPS SDS Running Buffer (20X)Thermo Fisher ScientificCatalog #NP0001
Tris-Glycine Transfer BufferInvitrogen - Thermo FisherCatalog #NP00061
VCP (7F3) Rabbit mAbCell Signaling TechnologyCatalog #2649
Anti-HaloTag(R) Monoclonal AntibodyPromegaCatalog #G9211
Procedures
Procedures
4h 57m 10s
4h 57m 10s
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 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.
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 1 mL growth media containing 50 nanomolar (nM) TMR-conjugated Halo ligand.
Note
- Aliquot the ligand into small aliquots and avoid freeze-and-thaw cycles.
- The ligand is sensitive to light so put the leftover away immediately after use and keep the media in the dark if you need to do a time course treatment.
Incubate in a normal TC incubator for 00:20:00 .
20m
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 2 mL of growth media containing 4 micromolar (µM) Antimycin A, 10 micromolar (µM) Oligomycin and 10 micromolar (µM) QVD for desired period.
Note
Make sure all drugs are vortexed well, mix the media well after adding each drug.
After the treatment, harvest the cells On ice by scraping.
Note
If you scan for fluorescence signal, from this step on consider keeping the samples away from light as much as possible.
Pre-chill eppies and 1x PBS On ice .
Note
I normally put all the plates that need harvesting into a fridge and harvest one by one On ice .
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.
Note
*Make sure swirl around after adding the PBS to wash the cells properly.
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 3000 x g, 4°C, 00:02:00 . Aspirate off PBS.
2m
Quickly centrifuge for 00:00:10 to spin down the residual PBS. Aspirate off all the PBS.
10s
Lyse the cell pellets in lysis buffer and boil the samples at 99 °C with shaking for 00:07:00 .
Note
I use the plastic clips to make sure that the lids won’t pop during heating.
7m
Let the samples cool down and spin at max speed (Room temperature ) for 00:01:00 .
1m
Estimate the protein concentration by nanodrop.
Note
Make sure the concentrations do not exceed 6 undetermined . If they do, dilute with lysis buffer and reheat them for a couple of minutes at 99 °C with shaking.
Aliquot 25 µg of each sample into a new eppie and add 1x LDS to make up to 15 µL .
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 00:10:00 and 190V for 00:55:00 .
1h 5m
Gels were then subjected to wet transfer onto PVDF membrane using cold NuPAGE transfer buffer containing 20% Methanol for 01:00:00 at Room temperature .
1h
After transfer,
Incubate PVDF membrane with PVDF destain buffer on a shaker at Room temperature for 00:02:00 .
2m
wash with PBS/Tween:
wash with PBS/Tween for 00:05:00 . (1/3)
wash with PBS/Tween for 00:05:00 . (2/3)
wash with PBS/Tween for 00:05:00 . (3/3)
15m
Block with blocking buffer for 00:15:00 .
15m
Remove blocking buffer,
Rinse:
Rinse with PBS/Tween. (1/2)
Rinse with PBS/Tween. (2/2)
Wash:
Wash with PBS/Tween for 00:05:00 . (1/2)
Wash with PBS/Tween for 00:05:00 . (2/2)
10m
Wash with 1x PBS for 00:05:00 .
5m
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 Overnight .
Note
To make sure we don’t lose antibodies, I wet the tubs with PBS/Tween before putting in the antibodies.
5m
The next day, recycle the antibodies back to their tubes,
Wash:
- wash the blots with PBS/Tween for00:05:00 . (1/3)
- wash the blots with PBS/Tween for00:05:00 . (2/3)
- wash the blots with PBS/Tween for00:05:00 . (3/3)
15m
Incubate with appropriate HRP-conjugated secondary antibodies (1/10000 for HALO and 1/5000 for VCP) made up in blocking buffer for 01:00:00 .
1h
Wash the blots and develop.
Wash the blots,
wash the blots with PBS/Tween for 00:05:00 . (1/2)
wash the blots with PBS/Tween for 00:05:00 . (2/2)
10m
Wash the blots with 1x PBS for 00:05:00 .
5m
Develop the blots with ECL prime.