Unbiased Millivolts Assay of Mitochondrial Membrane Potential in Intact Cells.
Bis-oxonol
Cell culture
Cellular heterogeneity
Fluorescence microscopy
Live cell microscopy
MitoTracker
Mitochondrial biogenesis
Mitochondrial membrane potential
Plasma membrane potential
Single cell
TMRM
Tetramethylrhodamine methyl ester
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
30
6
2022
pubmed:
1
7
2022
medline:
6
7
2022
Statut:
ppublish
Résumé
The mitochondrial membrane potential (ΔψM) is the major component of the bioenergetic driving force responsible for most cellular ATP produced, and it controls a host of biological processes. In intact cells, assay readouts with commonly used fluorescence ΔψM probes are distorted by factors other than ΔψM. Here, we describe a protocol to calculate both ΔψM and plasma membrane potential (ΔψP) in absolute millivolts in intact single cells, or in populations of adherent, cultured cells. Our approach generates unbiased data that allows comparison of ΔψM between cell types with different geometry and ΔψP, and to follow ΔψM in time when ΔψP fluctuates. The experimental paradigm results in fluorescence microscopy time courses using a pair of cationic and anionic probes with internal calibration points that are subsequently computationally converted to millivolts on an absolute scale. The assay is compatible with wide field, confocal or two-photon microscopy. The method given here is optimized for a multiplexed, partial 96-well microplate format to record ΔψP and ΔψM responses for three consecutive treatment additions.
Identifiants
pubmed: 35771433
doi: 10.1007/978-1-0716-2309-1_2
pmc: PMC9377305
mid: NIHMS1823917
doi:
Substances chimiques
Fluorescent Dyes
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
11-61Subventions
Organisme : NIA NIH HHS
ID : R01 AG055822
Pays : United States
Organisme : NIDA NIH HHS
ID : R41 DA043369
Pays : United States
Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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