Measurement of mitochondrial membrane potential was determined using the potential-sensitive dye Tetramethylrhodamine ethyl ester perchlorate (TMRE; Molecular Probes). Briefly, 2000-3000 cells were cultured in a 384-well microplate for 48 hours then incubated for 30 minutes in DMEM containing 20 nM MitoTracker Green FM (Molecular Probes). The cells were pre-incubated with 20 μM Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; to eliminate mitochondrial membrane potential) or solvent control at 37 °C for 1 hour prior to adding TMRE. After washing with media, 25 nM TMRE in media containing 0.5 μg/mL Hoechst 33342 (Molecular Probes) was added to the cells and incubated for 1 hour at 37 °C. TMRE and MitoTracker Green fluorescence intensities of the mitochondria in live cells were imaged using the Opera automated confocal microscope and analyzed with the Columbus Image Analysis Software (Perkin Elmer).
Mitotracker green
Manufactured by PerkinElmer
Sourced in Australia, United States
MitraTracker Green is a fluorescent dye used to visualize mitochondria in live cells. It is a cell-permeant, green-fluorescent stain that labels mitochondria. The dye accumulates in active mitochondria, allowing for the detection and quantification of mitochondrial mass and function.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker green fm, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Columbus image analysis software, by PerkinElmer (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Resazurin solution, by Biotium (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Operetta cls high content analysis system, by PerkinElmer (1 mentions)
Harmony high content imaging and analysis software, by PerkinElmer (1 mentions)
3 protocols using mitotracker green
1
Mitochondrial Membrane Potential Assay
2
Mitochondrial Activity Measurement in Gene-Silenced Cells
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HeLa cells were grown in Eagle’s Minimum Essential Medium (EMEM; GibcoBR) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA) 2 mM glutamine (Gibco, Waltham, MA, USA) and 1% penicillin-streptomycin (PS; 10,000 U/mL; Gibco). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2.
Ninety-six-well plates were used to assess mitochondrial activity and determine the number of mitochondria per well in gene-silenced cells.
Mitochondrial activity was determined by adding resazurin solution (10% vol/vol) (Biotium, Fremont, CA, USA) to culture medium, and relative quantification of mitochondria was performed with MitoTracker green FM at 150 nM in PBS (ThermoFisher Scientific, Waltham, MA, USA), following manufacturer’s indications. Resazurin reduction and MitoTracker green fluorescence emission were quantified using a Victor X5 fluorimeter (Perkin Elmer, Melbourne, Australia).
Both procedures display a direct linearity between 1000 and 10,000 cells per well.
Ninety-six-well plates were used to assess mitochondrial activity and determine the number of mitochondria per well in gene-silenced cells.
Mitochondrial activity was determined by adding resazurin solution (10% vol/vol) (Biotium, Fremont, CA, USA) to culture medium, and relative quantification of mitochondria was performed with MitoTracker green FM at 150 nM in PBS (ThermoFisher Scientific, Waltham, MA, USA), following manufacturer’s indications. Resazurin reduction and MitoTracker green fluorescence emission were quantified using a Victor X5 fluorimeter (Perkin Elmer, Melbourne, Australia).
Both procedures display a direct linearity between 1000 and 10,000 cells per well.
3
Mitochondrial and ROS Evaluation in hiPSC-CMs
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To evaluate mitochondrial membrane potential (MMP) and cellular ROS levels, hiPSC-CMs were cultured at 2×104/well density in matrigel-coating 96-well plate with optical optimization bottom (PerkinElmer, USA). The cells were incubated with different concentrations of ZnO NPs for 6 h. Afterward, the cells were washed with PBS and labeled with co-staining include: I. nucleus probe Hoechst 33342 (1 μM), intracellular ROS probe CM-H2DCFDA (10 μM), and MMP probe TMRM (100 nM) for 15 min at 37°C; II. nuclei probe Hoechst 33342 (1 μM) and mitochondria content probe Mito-Tracker Green (100 nM) for 30 min at 37°C. All above-mentioned probes were purchased from Invitrogen (USA) and dissolved in culture medium. Cells were then washed twice with PBS to thoroughly remove the fluorescent probes. Operetta CLS™ high-content analysis system (PerkinElmer, USA) was used to capture images at specific fluorescence excitation/emission wavelength according to probe instructions (Hoechst 33342, 350/461 nm; CM-H2DCFDA, 485/530 nm, TMRM 548/574 nm, Mito-Tracker Green 490/516 nm). Each group had at least three replicate wells, and at least 9 fields of view were randomly selected for quantitative fluorescence analysis. The fluorescence intensity of each probe within the cells was performed automatically by Harmony® high-content imaging and analysis software (PerkinElmer, USA; version 4.1).
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