Mitochondrial membrane potential (ΔΨ) was determined using JC-10™ (AAT Bioquest, Sunnyvale, CA), a cationic and lipophilic dye that selectively enters mitochondria. The mitochondrial uptake of JC-10™ is directly associated with ΔΨ. In general, JC-10™ accumulates in the mitochondrial matrix where it forms red fluorescent aggregates. In uncoupled cells, JC-10™ diffuses out of the mitochondria and changes to monomeric forms and stains the cell a green fluorescence. The primary neuronal cells were suspended in 1 mL culture medium with JC-10™ (7.5 μM) for 30 min at 37 °C, 5% CO2. The JC-10™ stained cells were centrifuged (1200×g, 5 min) and the pelleted cells were washed three times with 1 mL PBS containing FBS (3%). The stained cells were resuspended in PBS. The fluorescent intensities for aggregate and monomeric forms of JC-10™ were measured at Ex/Em = 490/525 nm (FITC channel) and 540/595 nm (TRITC channel) in triplicate using 200 μL of stained cell suspension in a 96-well fluorometric plate reader (SpectraMax i3, Molecular Devices). The aggregate/monomer ratio was calculated and was proportional to the mitochondrial membrane potential 108 (link).
Jc 10
Manufactured by AAT Bioquest
Sourced in United States
The JC-10 is a compact, reliable, and easy-to-use spectrofluorometer designed for accurate fluorescence measurements. It features a high-intensity LED light source, monochromator-based optics, and a sensitive photodetector to provide precise excitation and emission wavelength selection. The JC-10 is a versatile instrument suitable for a wide range of fluorescence-based applications in research and analytical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Pherastar fsx, by BMG Labtech (3 mentions)
Jc 10, by Merck Group (3 mentions)
Carbonyl cyanide 3 chlorophenylhydrazone cccp, by Merck Group (2 mentions)
Jc 10, by Molecular Devices (1 mentions)
Spectramax i3, by Molecular Devices (1 mentions)
Jc 10, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Spark multimode microplate reader, by Tecan (1 mentions)
Facscalibur system, by BD (1 mentions)
Metaxpress software, by Molecular Devices (1 mentions)
Hoechst 33342, by AAT Bioquest (1 mentions)
9 protocols using jc 10
1
Mitochondrial Membrane Potential Analysis
2
Mitochondrial Membrane Potential Analysis
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For measuring the mitochondrial membrane potential, the JC-10 from AAT Bioquest (Sunnyvale, CA, USA) was used. Cells were cultivated in black 96-well plates for 6 weeks. Cells were washed with Hank’s balanced salt solution (HBSS) and incubated with 20 µM JC-10 in HBSS (Thermo Fisher Scientific, Waltham, MA, USA)/EXS-HEPES (1:1) for 45 min, and the fluorescence intensity at Ex/Em = 490/525 nm and 540/590 nm was measured with a Spark® multimode microplate reader (TECAN, Hamburg, Germany). Graphs show the calculated ratio of Em 590/525.
3
Mitochondrial Membrane Potential Analysis
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JC-10 enters the mitochondria, where it is retained in multimeric form while the compartment remains polarized. During membrane depolarization the JC-10 is converted to the monomeric form. Briefly, JC-10 (AAT Bioquest) was added to cells coated onto a black microtiter plate. Cells were then stimulated as above in the presence of JC-10. After 5 min, the fluorescence of the multimeric form (Ex540/590) and monomeric form (Ex490Em525) were measured. Results were expressed using the ratio of 525/590.
4
Quantifying Mitochondrial Membrane Potential
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To measure mitochondrial membrane potential (ΔΨm), cells were washed with Krebs buffer (145 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM MgCl2, 1 mM CaCl2, 5.6 mM glucose and pH 7.4/NaOH) and loaded with JC-10 (5 μM; AAT Bioquest, Inc.) at 37°C for 30 min. Green fluorescence (depolarization) was monitored at 529 nm and red fluorescence (polarized) at 590 nm on a multi-mode plate reader PHERAstar FSX (BMG Labtech). To establish that the JC-10 signal was indicative of ΔΨm, experiments were terminated inducing a maximal mitochondrial depolarization by addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP 10 μM; Sigma-Aldrich).
5
Mitochondrial Membrane Potential Measurement
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To detect changes in mitochondrial membrane potential (ΔΨm), cells were washed with Krebs buffer (145 mm NaCl, 5 mm KCl, 10 mm HEPES, 1 mm MgCl2, 1 mm CaCl2, 5.6 mm glucose and pH 7.4/NaOH) and loaded with 5 μm JC-10 (a derivative of 5,5′,6,6′-tetrachloro-1,1,3,3′-tetraethyl-benzimidazolyl-carbocyanine iodide, JC-1; AAT Bioquest, Inc.) at 37°C for 30 min. The fluorescence intensities were measured using the multi-mode plate reader PHERAstar FSX (BMG Labtech) by fluorescence excitation/emission maxima: 514/529 nm, monomer form, and 585/590 nm, aggregate form. To establish that the JC-10 signal was indicative of ΔΨm, experiments were terminated inducing a maximal mitochondrial depolarization by addition of 10 μm carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma-Aldrich).
6
Measuring Mitochondrial Membrane Potential
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The mitochondrial membrane potentials of cultured cells were determined by using the fluorescent probe JC-10 (AAT Bioquest, Sunnyvale, CA, USA) following the manufacturer's recommendations. Briefly, cultured cells were exposed to 5 μM mitochondrial uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) for 1 hr and then incubated in culture medium containing JC-10 for 1 hr at room temperature. The cells were washed with PBS and analysed by flow cytometry. Photomultiplier settings were adjusted to detect JC-10 monomer and aggregate fluorescence on the FL1 (525 nm) and FL2 (595 nm) detectors. The fluorescence ratio at these wavelengths was used to monitor changes in mitochondrial membrane potential.
7
Mitochondrial Membrane Potential Assessment
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To detect changes in mitochondrial membrane potential (ΔΨm), cells were incubated with 5 μm JC-10 (AAT Bioquest, Inc.) at 37°C for 1 h. The fluorescence intensities were measured using the multi-mode plate reader PHERAstar FSX (BMG Labtech) by fluorescence excitation/emission maxima: 514/529 nm, monomer form, and 585/590 nm, aggregate form. To establish that the JC-10 signal was indicative of ΔΨm, experiments were terminated inducing a maximal mitochondrial depolarization by addition of 10 μm carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Merck).
8
Fluorescence Analysis of JC-10 Stained Cells
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The culture medium was changed to F-12 and was supplemented with 15 μM JC-10 (AAT Bioquest, Sunnyvale, CA, USA). After incubation was performed at 37 °C for 30 min, fluorescence was investigated using the BD FACSCalibur system.
9
Cytotoxicity and Mitochondrial Integrity Assay
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