Aug 09, 2023

Public workspaceMeasuring mitophagy via FACS with mtKeima reporter

DOI
dx.doi.org/10.17504/protocols.io.5qpvo3rd9v4o/v1
  • 1Lazarou Lab, WEHI
Louise Uoselis
Open access
DOI: dx.doi.org/10.17504/protocols.io.5qpvo3rd9v4o/v1
Protocol Citation: Louise Uoselis 2023. Measuring mitophagy via FACS with mtKeima reporter. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3rd9v4o/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: August 09, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86216
Keywords: ASAPCRN
Abstract
Preparation of samples for measuring mitophagy levels using mtKeima reporter by fluorescence activated cell sorting (FACS).
Day 1
Day 1
Seed cells in a 24 well plate, aiming for a confluency of ~80-90% at the time of treatment. Seed additional wells of cells not expressing any fluorescent proteins, cells expressing only mtKeima, and cells expressing only YFP-Parkin (to be used as gating controls).
Day 2
Day 2
2h 3m
2h 3m
Feed all cells with standard growth media for Duration01:00:00 prior to treatment.

1h
Replace media in each well with media containing the drug you are treating with.
NOTE: Do not change the media or treat the additional wells of cells to be used for gating control.
Duration01:00:00 prior to harvesting, feed the untreated wells with Amount0.5 mL of fresh growth media.

1h
At the conclusion of the treatment time point, harvest the cells using the following procedure:
Aspirate media from all wells.
Wash all wells once with Amount500 µL of TemperatureRoom temperature PBS.

Add Amount150 µL of trypsin to each well, and incubate cells at Temperature37 °C for Duration00:01:30

1m 30s
Place plates onto ice, and harvest each sample into a separate microfuge tube on ice by resuspending each sample with Amount500 µL of ice cold standard growth media.

Centrifuge all samples at Centrifigation1000 rcf for Duration00:01:30 at Temperature4 °C

1m 30s
Carefully aspirate the supernatant from all samples.
Resuspend each sample in Amount50 µL of FACS sorting media (10% v/v FBS, 1 mM EDTA in PBS) and place into FACS analysis tubes. Keep samples on ice until immediately prior to analysis.