Mitochondrial membrane-potential and mitochondrial mass staining
Materials Required
Principle
Mitochondrial membrane potential staining measures the electrochemical polarization across the mitochondrial inner membrane in live cells using lipophilic cationic fluorescent probes; early rhodamine-based work showed that selective mitochondrial dye accumulation is lost when the mitochondrial transmembrane potential is dissipated[1][2][3][4]. JC-1 reports mitochondrial polarization by shifting from green monomer fluorescence to red J-aggregate fluorescence as dye concentration increases within energized mitochondria; therefore, the red/green fluorescence ratio is used as a relative readout of mitochondrial membrane potential[5][6][7][8]. TMRE or TMRM staining provides a single-channel relative readout because these cationic rhodamine esters accumulate in polarized mitochondria, and lower fluorescence indicates reduced mitochondrial polarization when acquisition and dye-loading conditions are controlled[3][4][9]. Mitochondrial mass staining is commonly performed with MitoTracker Green FM or related MitoTracker dyes as a semi-quantitative flow-cytometry or imaging readout of mitochondrial content, but published studies emphasize that dye concentration, cell type, mitochondrial potential, transporter activity, and analysis strategy can affect interpretation[10][11][12][13].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use live-cell-compatible culture medium or PBS-based staining buffer for dye incubation and washing, because JC-1, TMRE/TMRM, and MitoTracker-based assays are designed for viable-cell staining rather than fixed-cell loading[4][7][9][11].
• Use DMSO only as a dye solvent when preparing concentrated stocks, and include a vehicle-stained control when comparing treated and untreated samples[7][9].
• Use FCCP or CCCP as a depolarization control when validating mitochondrial membrane-potential staining, because uncouplers collapse mitochondrial polarization and reduce potential-dependent mitochondrial dye retention or JC-1 aggregate formation[2][4][7][9].
• Use JC-1 when a ratiometric mitochondrial membrane-potential readout is required; published protocols report 2 µM JC-1 for 15-30 min at 37 °C and read green and red fluorescence after staining[6][7].
• Use TMRE or TMRM when a single-channel mitochondrial membrane-potential readout is preferred; published protocols and methods papers use low nanomolar working concentrations and interpret fluorescence intensity as a relative membrane-potential signal[3][4][9][14].
• Use MitoTracker Green FM for mitochondrial mass or mitochondrial-content staining; published flow-cytometry and imaging-flow-cytometry methods use it as a semi-quantitative mitochondrial-content probe and stress that staining concentration should be optimized for the cell type and instrument[10][11][12][13].
• Optional viability dye may be included when compatible with the fluorescence panel, because dead or damaged cells can distort live-cell mitochondrial dye measurements[4][11].
• Use a fluorescence microscope, confocal microscope, plate reader, conventional flow cytometer, or imaging flow cytometer according to the required readout; JC-1 has been measured by microscopy and flow cytometry, TMRE/TMRM by flow cytometry or microscopy, and MitoTracker Green by flow cytometry or imaging flow cytometry[5][6][7][9][11][12].
• For JC-1 flow cytometry, use green and red detection channels and consider 405 nm excitation for the aggregate signal when available, because 405 nm excitation was reported to reduce monomer spillover into the aggregate measurement compared with standard 488 nm excitation[5].
• Use DMSO only as a dye solvent when preparing concentrated stocks, and include a vehicle-stained control when comparing treated and untreated samples[7][9].
• Use FCCP or CCCP as a depolarization control when validating mitochondrial membrane-potential staining, because uncouplers collapse mitochondrial polarization and reduce potential-dependent mitochondrial dye retention or JC-1 aggregate formation[2][4][7][9].
• Use JC-1 when a ratiometric mitochondrial membrane-potential readout is required; published protocols report 2 µM JC-1 for 15-30 min at 37 °C and read green and red fluorescence after staining[6][7].
• Use TMRE or TMRM when a single-channel mitochondrial membrane-potential readout is preferred; published protocols and methods papers use low nanomolar working concentrations and interpret fluorescence intensity as a relative membrane-potential signal[3][4][9][14].
• Use MitoTracker Green FM for mitochondrial mass or mitochondrial-content staining; published flow-cytometry and imaging-flow-cytometry methods use it as a semi-quantitative mitochondrial-content probe and stress that staining concentration should be optimized for the cell type and instrument[10][11][12][13].
• Optional viability dye may be included when compatible with the fluorescence panel, because dead or damaged cells can distort live-cell mitochondrial dye measurements[4][11].
• Use a fluorescence microscope, confocal microscope, plate reader, conventional flow cytometer, or imaging flow cytometer according to the required readout; JC-1 has been measured by microscopy and flow cytometry, TMRE/TMRM by flow cytometry or microscopy, and MitoTracker Green by flow cytometry or imaging flow cytometry[5][6][7][9][11][12].
• For JC-1 flow cytometry, use green and red detection channels and consider 405 nm excitation for the aggregate signal when available, because 405 nm excitation was reported to reduce monomer spillover into the aggregate measurement compared with standard 488 nm excitation[5].
Experimental Procedure
• Prepare live single-cell suspensions or live adherent-cell cultures immediately before staining, because the cited membrane-potential dyes are interpreted in viable cells and potential-dependent signals can change during sample handling[4][7][9].
• Prepare unstained, single-dye, vehicle, and uncoupler-treated controls before acquisition; uncoupler controls define the depolarized signal range, while unstained and single-dye controls support background subtraction, compensation, or spectral separation[4][5][7][9].
• For mitochondrial mass staining, perform a dye-titration pilot in the relevant cell type before the main experiment, because imaging-flow-cytometry and flow-cytometry studies showed that MitoTracker dye concentration and analysis gates influence false-positive and false-negative mitochondrial-content measurements[11][12].
• For JC-1 staining, incubate live cells with 2 µM JC-1 for 15-30 min at 37 °C, protect samples from light, wash or resuspend according to the acquisition format, and measure green monomer fluorescence and red aggregate fluorescence by microscopy, plate reader, or flow cytometry[6][7].
• For JC-1 positive depolarization control, treat a matched sample with FCCP or CCCP before or during the assay according to the chosen published protocol, then confirm reduced red/green JC-1 ratio relative to vehicle control[4][6][7].
• For TMRE/TMRM staining, incubate live cells with the selected low-nanomolar dye concentration under matched conditions for all samples, acquire fluorescence promptly, and interpret decreased TMRE/TMRM fluorescence as reduced relative mitochondrial polarization only after comparison with vehicle and depolarized controls[3][4][9][14].
• For mitochondrial mass staining, incubate live cells with MitoTracker Green FM under the optimized concentration for the cell type and instrument, then quantify mitochondrial-content signal by flow cytometry, imaging flow cytometry, or microscopy using the same acquisition settings across experimental groups[10][11][12][13].
• When measuring both mitochondrial membrane potential and mitochondrial mass, stain matched samples or compatible co-stained samples and analyze membrane-potential fluorescence with mitochondrial-content signal so that changes in potential are not mistaken for changes in mitochondrial abundance[4][10][11][13].
• For JC-1, calculate the red/green fluorescence ratio per sample or per cell population; a reduced red/green ratio indicates lower relative mitochondrial membrane potential, while unchanged green or red signal alone should not be interpreted without the ratio and controls[5][6][7][8].
• For TMRE/TMRM, report median or mean fluorescence intensity in live, single cells and compare it with vehicle and uncoupler-treated controls; because TMRE/TMRM are single-channel probes, the result is a relative fluorescence-based membrane-potential measurement rather than an absolute voltage unless a separate quantitative calibration method is used[3][4][9][14].
• For MitoTracker Green-based mitochondrial mass, report median or mean fluorescence intensity after excluding debris, doublets, and dead cells, and present the analysis gate consistently across samples; T-cell studies specifically show that objective gating and optimized dye concentration improve reproducibility[11][12].
• Use biological replicates and statistical tests appropriate to the experimental design, and avoid pooling technical replicates as independent biological observations[11][12].
• Prepare unstained, single-dye, vehicle, and uncoupler-treated controls before acquisition; uncoupler controls define the depolarized signal range, while unstained and single-dye controls support background subtraction, compensation, or spectral separation[4][5][7][9].
• For mitochondrial mass staining, perform a dye-titration pilot in the relevant cell type before the main experiment, because imaging-flow-cytometry and flow-cytometry studies showed that MitoTracker dye concentration and analysis gates influence false-positive and false-negative mitochondrial-content measurements[11][12].
• For JC-1 staining, incubate live cells with 2 µM JC-1 for 15-30 min at 37 °C, protect samples from light, wash or resuspend according to the acquisition format, and measure green monomer fluorescence and red aggregate fluorescence by microscopy, plate reader, or flow cytometry[6][7].
• For JC-1 positive depolarization control, treat a matched sample with FCCP or CCCP before or during the assay according to the chosen published protocol, then confirm reduced red/green JC-1 ratio relative to vehicle control[4][6][7].
• For TMRE/TMRM staining, incubate live cells with the selected low-nanomolar dye concentration under matched conditions for all samples, acquire fluorescence promptly, and interpret decreased TMRE/TMRM fluorescence as reduced relative mitochondrial polarization only after comparison with vehicle and depolarized controls[3][4][9][14].
• For mitochondrial mass staining, incubate live cells with MitoTracker Green FM under the optimized concentration for the cell type and instrument, then quantify mitochondrial-content signal by flow cytometry, imaging flow cytometry, or microscopy using the same acquisition settings across experimental groups[10][11][12][13].
• When measuring both mitochondrial membrane potential and mitochondrial mass, stain matched samples or compatible co-stained samples and analyze membrane-potential fluorescence with mitochondrial-content signal so that changes in potential are not mistaken for changes in mitochondrial abundance[4][10][11][13].
• For JC-1, calculate the red/green fluorescence ratio per sample or per cell population; a reduced red/green ratio indicates lower relative mitochondrial membrane potential, while unchanged green or red signal alone should not be interpreted without the ratio and controls[5][6][7][8].
• For TMRE/TMRM, report median or mean fluorescence intensity in live, single cells and compare it with vehicle and uncoupler-treated controls; because TMRE/TMRM are single-channel probes, the result is a relative fluorescence-based membrane-potential measurement rather than an absolute voltage unless a separate quantitative calibration method is used[3][4][9][14].
• For MitoTracker Green-based mitochondrial mass, report median or mean fluorescence intensity after excluding debris, doublets, and dead cells, and present the analysis gate consistently across samples; T-cell studies specifically show that objective gating and optimized dye concentration improve reproducibility[11][12].
• Use biological replicates and statistical tests appropriate to the experimental design, and avoid pooling technical replicates as independent biological observations[11][12].
Troubleshooting
Problem: JC-1 red aggregate signal is weak in all samples.
• Possible Cause: Cells may be depolarized, overloaded, damaged, or acquired with suboptimal excitation/emission settings.• Literature-supported Solution: Confirm viability, include vehicle and FCCP/CCCP controls, use the reported 2 µM JC-1 15-30 min 37 °C staining window, and consider 405 nm excitation for the aggregate channel if available[5][6][7].
Problem: JC-1 signal differs between cell lines despite similar mitochondrial biology.
• Possible Cause: ABCB1/P-glycoprotein activity can reduce intracellular JC-1 accumulation and suppress aggregate formation.• Literature-supported Solution: Avoid comparing JC-1 readouts across transporter-positive and transporter-negative cells without transporter-status controls; Elefantová et al. showed that tariquidar restored JC-1 accumulation in P-glycoprotein-positive L1210 cells, whereas verapamil and cyclosporine A did not fully restore the signal[8].
Problem: MitoTracker Green signal does not match expected mitochondrial content.
• Possible Cause: Dye concentration, cell type, and analysis strategy can produce false-positive or false-negative mitochondrial-content measurements.• Literature-supported Solution: Perform dye titration, keep acquisition settings constant, and use objective gating or image-based validation when possible[11][12].
Problem: TMRE/TMRM fluorescence changes after treatment but mitochondrial mass also changes.
• Possible Cause: Single-channel potential dyes reflect relative dye accumulation and can be confounded by altered mitochondrial content.• Literature-supported Solution: Measure mitochondrial mass in parallel with MitoTracker Green or another mitochondrial-content readout and interpret potential signal relative to mitochondrial-content signal[3][4][10][13].
References:
- [1]. Johnson LV, et al. Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci U S A. 1980;77(2):990-994. [Content Brief]
- [2]. Johnson LV, et al. Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J Cell Biol. 1981;88(3):526-535. [Content Brief]
- [3]. Scaduto RC Jr, et al. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J. 1999;76(1 Pt 1):469-477. [Content Brief]
- [4]. Perry SW, et al. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques. 2011;50(2):98-115. [Content Brief]
- [5]. Perelman A, et al. JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry. Cell Death Dis. 2012;3:e430. [Content Brief]
- [6]. Chen G, Yang Y, Xu J, et al. A flow cytometry-based assay for measuring mitochondrial membrane potential in cardiac myocytes after hypoxia/reoxygenation. J Vis Exp. 2018;(137):57725. [Content Brief]
- [7]. Sivandzade F, et al. Analysis of the mitochondrial membrane potential using the cationic JC-1 dye as a sensitive fluorescent probe. Bio Protoc. 2019;9(1):e3128. [Content Brief]
- [8]. Elefantova K, et al. Detection of the mitochondrial membrane potential by the cationic dye JC-1 in L1210 cells with massive overexpression of the plasma membrane ABCB1 drug transporter. Int J Mol Sci. 2018;19(7):1985. [Content Brief]
- [9]. Crowley LC, et al. Measuring mitochondrial transmembrane potential by TMRE staining. Cold Spring Harb Protoc. 2016;2016(12). [Content Brief]
- [10]. Pendergrass W, et al. Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry A. 2004;61(2):162-169. [Content Brief]
- [11]. Gautam N, et al. A high content imaging flow cytometry approach to study mitochondria in T cells: MitoTracker Green FM dye concentration optimization. Methods. 2018;134-135:11-19. [Content Brief]
- [12]. Clutton G, et al. A reproducible, objective method using MitoTracker fluorescent dyes to assess mitochondrial mass in T cells by flow cytometry. Cytometry A. 2019;95(4):450-456. [Content Brief]
- [13]. Girotra M, Ritchie B, Stone L, et al. Measurement of mitochondrial mass and membrane potential in hematopoietic stem cells and T-cells by flow cytometry. J Vis Exp. 2019;(154):e60475. [Content Brief]
- [14]. Cottet-Rousselle C, et al. Cytometric assessment of mitochondria using fluorescent probes. Cytometry A. 2011;79(6):405-425. [Content Brief]
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