Sep 21, 2022
Live-cell imaging: Mitochondria membrane potential
- 1UCL Institute of Neurology
Protocol Citation: gurvir.virdi, mineechoi 2022. Live-cell imaging: Mitochondria membrane potential. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwj997lmk/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: September 21, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70338
Keywords: ASAPCRN
Abstract
Mitochondrial membrane potential is the electrogenic potential between the inner membrane and matrix of mitochondria which, in combination with the mitochondrial pH gradient, provides the force to drive protons into mitochondria to generate ATP.
This protocol presents instructions on how to measure mitochondrial membrane potential in live-cell imaging.
Cells were washed 2x with HBSS (Invitrogen)
They are then incubated w 25 nM tetramethylrhodamine methyl ester (TMRM, Thermo Fisher Scientific) in HBSS for 00:40:00 at Room temperature.
Note
TMRM is a lipophilic cationic dye that accumulates within mitochondria in inverse proportion to mitochondrial membrane potential (Δψm) according to the Nernst equation.
40m
After 40 minutes, confocal microscope imaging is performed in the presence of TMRM (in HBSS); the 560 nm laser was used to excite, and fluorescence was measured above 580 nm.
Live-cell imaging is performed using a confocal microscope (Zeiss LSM 710 or 880 with an integrated META detection system). For confocal microscopes, illumination intensity is limited to 0.1-0.2% of laser output to prevent phototoxicity, and the pinhole is set to allow optical slice at approximately 1-2 μm. Z-stack images are collected.
For dynamic TMRM a time-series with 5 second intervals was performed on the same confocal microscopy set up.