Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Brand MD (1995) Measurement of mitochondrial protonmotive force. In: Brown GC, Cooper CE (eds) Bioenergetics: a practical approach. IRL Press, Oxford, pp 39–62
Sekine S, Youle RJ (2018) PINK1 import regulation; a fine system to convey mitochondrial stress to the cytosol. BMC Biol 16:1–12. https://doi.org/10.1186/s12915-017-0470-7
Schendzielorz AB, Schulz C, Lytovchenko O et al (2017) Two distinct membrane potential-dependent steps drive mitochondrial matrix protein translocation. J Cell Biol 216:83–92. https://doi.org/10.1083/jcb.201607066
Palmieri F (2013) The mitochondrial transporter family SLC25: identification, properties and physiopathology. Mol Asp Med 34:465–484. https://doi.org/10.1016/j.mam.2012.05.005
Kim SJ, Xiao J, Wan J et al (2017) Mitochondrially derived peptides as novel regulators of metabolism. J Physiol 595:6613–6621. https://doi.org/10.1113/JP274472
Nicholls DG (2012) Fluorescence measurement of mitochondrial membrane potential changes in cultured cells. Methods Mol Biol 810:119–133
Zorova LD, Popkov VA, Plotnikov EY et al (2018) Mitochondrial membrane potential. Anal Biochem 552:50–59. https://doi.org/10.1016/j.ab.2017.07.009
Lemasters JJ, Ramshesh VK (2007) Imaging of mitochondrial polarization and depolarization with cationic fluorophores. Methods Cell Biol 80:283–295
Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435:297–312. https://doi.org/10.1042/BJ20110162
Choi SW, Gerencser AA, Nicholls DG (2009) Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure. J Neurochem 109:1179–1191. https://doi.org/10.1111/j.1471-4159.2009.06055.x
Merlini L, Angelin A, Tiepolo T et al (2008) Cyclosporin a corrects mitochondrial dysfunction and muscle apoptosis in patients with collagen VI myopathies. Proc Natl Acad Sci U S A 105:5225–5229. https://doi.org/10.1073/pnas.0800962105
Gerencser AA, Chinopoulos C, Birket MJ et al (2012) Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria. J Physiol 590:2845–2871. https://doi.org/10.1113/jphysiol.2012.228387
Keij JF, Bell-Prince C, Steinkamp JA (2000) Staining of mitochondrial membranes with 10-nonyl acridine orange, MitoFluor green, and MitoTracker green is affected by mitochondrial membrane potential altering drugs. Cytometry 39:203–210. https://doi.org/10.1002/(sici)1097-0320(20000301)39:3<203::aid-cyto5>3.0.co;2-z
Kholmukhamedov A, Schwartz JM, Lemasters JJ (2013) Isolated mitochondria infusion mitigates ischemia-reperfusion injury of the liver in rats: mitotracker probes and mitochondrial membrane potential. Shock 39:543. https://doi.org/10.1097/01.shk.0000430660.63077.7f
Gerencser AA, Mookerjee SA, Jastroch M, Brand MD (2016) Measurement of the absolute magnitude and time courses of mitochondrial membrane potential in primary and clonal pancreatic beta-cells. PLoS One 11:e0159199. https://doi.org/10.1371/journal.pone.0159199
Nicholls DG (2006) Simultaneous monitoring of Ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons. J Biol Chem 281:14864–14874
Gerencser AA (2015) Bioenergetic analysis of single pancreatic β-cells indicates an impaired metabolic signature in type 2 diabetic subjects. Endocrinology 156:3496–3503. https://doi.org/10.1210/en.2015-1552
Gerencser AA (2018) Metabolic activation-driven mitochondrial hyperpolarization predicts insulin secretion in human pancreatic beta-cells. Biochim Biophys Acta Bioenerg 1859:817–828. https://doi.org/10.1016/j.bbabio.2018.06.006
Gerencser AA, Mookerjee SA, Jastroch M, Brand MD (2017) Positive feedback amplifies the response of mitochondrial membrane potential to glucose concentration in clonal pancreatic beta cells. Biochim Biophys Acta Mol basis Dis 1863:1054–1065. https://doi.org/10.1016/j.bbadis.2016.10.015
Birket MJ, Orr AL, Gerencser AA et al (2011) A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells. J Cell Sci 124:348–358. https://doi.org/10.1242/jcs.072272
Guntur AR, Gerencser AA, Le PT et al (2018) Osteoblast-like MC3T3-E1 cells prefer glycolysis for ATP production but adipocyte-like 3T3-L1 cells prefer oxidative phosphorylation. J Bone Miner Res 33:1052–1065. https://doi.org/10.1002/jbmr.3390
Rovini A, Heslop K, Hunt EG et al (2021) Quantitative analysis of mitochondrial membrane potential heterogeneity in unsynchronized and synchronized cancer cells. FASEB J 35:100–106. https://doi.org/10.1096/fj.202001693R
Nicholls DG, Ward MW (2000) Mitochondrial membrane potential and cell death: mortality and millivolts. Trends Neurosci 23:166–174. https://doi.org/10.1016/S0166-2236(99)01534-9
Acknowledgments
The authors thank Kareem Heslop (Hollings Cancer Center, Medical University of South Carolina, SC. USA) for critical reading of the manuscript, Christopher Wiley and Leyla Teos (Buck Institute for Research on Aging, CA, USA) for providing cultures of BJ1 cells and freshly isolated cardiomyocytes, respectively. This work was supported by the National Institutes of Health (NIH) grants 1R41DA043369 to AAG and R01AG055822 to Simon Melov. AAG declares a financial interest in Image Analyst Software.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
1 Electronic Supplementary Material
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Lerner, C.A., Gerencser, A.A. (2022). Unbiased Millivolts Assay of Mitochondrial Membrane Potential in Intact Cells. In: Tomar, N. (eds) Mitochondria. Methods in Molecular Biology, vol 2497. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2309-1_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2309-1_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2308-4
Online ISBN: 978-1-0716-2309-1
eBook Packages: Springer Protocols