May 24, 2023

Mito-Keima assay to assess mitophagy

DOI
https://dx.doi.org/10.17504/protocols.io.q26g74e1qgwz/v1
  • Thanh Ngoc Nguyen1
  • 1Laboratory of Michael Lazarou, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
  • ASAP workspace
nguyen.tha
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g74e1qgwz/v1
Protocol Citation: Thanh Ngoc Nguyen 2023. Mito-Keima assay to assess mitophagy. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74e1qgwz/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: July 18, 2022
Last Modified: May 31, 2024
Protocol  Integer ID: 67007
Keywords: Mito-Keima, Mitophagy, BFP-Parkin, ASAPCRN, mitophagy, procedure of mito, mitophagy this protocol, mito
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000350
Abstract
This protocol details the procedure of mito-keima assay to assess mitophagy.
Attachments
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Materials
Growth media:

DMEM:
AB
FBS10%
Glucose4.5 g/l
GlutaMAXTM1x
MEM NEAA1x
HEPES25 mM
  • 45% D-( )-GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8769
  • GlutaMAX™ SupplementThermo Fisher ScientificCatalog #35050061
  • MEAA (MEM Non-Essential Amino Acids)Gibco - Thermo Fisher ScientificCatalog #11140050
  • Antimycin A from Streptomyces sp.Merck MilliporeSigma (Sigma-Aldrich)Catalog #A8674 (made up in 100% Ethanol to 20 mg/ml)
  • Oligomycin (Calbiochem, 495455; made up in DMSO to 10 mg/ml) and
  • qVDMedChemExpressCatalog #HY-12305 (made up in DMSO to 10 mM)
  • 1x PBS
  • FACS buffer: 10% FBS, 0.5 millimolar (mM) EDTA in 1xPBS

Procedure
2h 9m
Seed the HeLa cells the day before the treatment day in 24 well plates.
Note
Each well contained 0.5 ml of growth media; 120,000 cells were seeded for penta KO expressing BFP-Parkin, mito-Keima (mtKeima) and GFP-OPTN or -NDP52; the number of cells of other cell lines were adjusted so that the next day they are all in similar confluency with penta KO expressing BFP-Parkin, mtKeima and GFP-OPTN or -NDP52.

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.
Note
Not concentrated in the middle of the well because this can affect the results.

Aspirate off the old media and treat each well with 0.5 mL of growth media containing 4 micromolar (µM) Antimycin A, 10 micromolar (µM) Oligomycin and 10 micromolar (µM) QVD for indicated times.
Note
Make sure all drugs are vortexed well, mix the media well after adding each drug.

Treat the longest time points first.
2 hours prior to harvesting, feed the untreated wells with 1 mL 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 0.5 mL of 1x PBS.
Note
Make sure swirl around after adding the PBS to wash the cells properly.

Aspirate 1x PBS and add 150 µL of trypsin and incubate at 37 °C for 00:02:00 -00:05:00 .
Note
Check under microscope to make sure all the cells were trypsinised properly.

7m
Add 300 µL of growth media to each well.
Mix well with a P1000 and transfer the cells to cold eppies.
Centrifuge the eppies at 1000 x g for 00:02:00 at 4 °C .
2m
Aspirate off the liquid.
Resuspend the cell pellets in 150 µL of FACS buffer, transfer to pre-chilled FACS tubes and keep the samples On ice in the dark for analysis.