Mitochondrial membrane potential (Ψm) was determined using an anionic (lipophilic) mitochondrial JC-1 dye(5, 5′, 6,6′-tetrachloro1, 1′, 3, 3′tetraethylbenzimidozolylcarbocyanine iodide) (Invitrogen, T-3168) as described previously41 (link). In brief, cells were washed using 1XPBS and incubated for 15 min at 37 °C with media containing 10 μM JC-1 dye. Emitted green (535 nm) and red (595 nm) fluorescence intensities were analyzed by filter-based multi-mode microplate reader (Clariostar, BMG Labtech, Germany) and ratiometric comparative fluorescence of the two were plotted using graph pad prism 5.1.
T3168
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The T3168 is a laboratory equipment product designed for general laboratory use. It serves as a versatile tool for various applications within a research or analytical setting. The core function of the T3168 is to provide a reliable and consistent performance in laboratory environments. However, a more detailed and unbiased description cannot be provided while maintaining the requested level of conciseness and objectivity.
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Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
96 well black flat bottom plate, by Corning (2 mentions)
Spectramax m2, by Molecular Devices (2 mentions)
Lab tek 2 chamber, by Thermo Fisher Scientific (2 mentions)
Dulbecco s phosphate buffer saline pbs, by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Clariostar, by BMG Labtech (1 mentions)
Guava easy cyte 8, by Merck Group (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Fl amg, by Thermo Fisher Scientific (1 mentions)
A1372, by Thermo Fisher Scientific (1 mentions)
Facscalibur system, by BD (1 mentions)
Jc 1 dye, by Thermo Fisher Scientific (1 mentions)
Infinite m200, by Tecan (1 mentions)
Jc 1 fluorescence dye, by Thermo Fisher Scientific (1 mentions)
Lsm 810, by Zeiss (1 mentions)
Facsverse, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Fluorometer, by Agilent Technologies (1 mentions)
27 protocols using t3168
1
Mitochondrial Membrane Potential Measurement
2
Mitochondrial Membrane Potential Assay
3
Mitochondrial Membrane Potential Assay in H9c2 Cells
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H9c2 cells were seeded on to 96-well black flat bottom plates (Costar). After 24 hours, cells were incubated in DMEM media without FBS and phenol red at 37 °C/5% CO2 with 5 μM rotenone (inhibits complex I) or 30 μM antimycin A (inhibits complex III) for 24 hours. In some experiments, cells were also incubated with 250 μM trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP, uncoupler of membrane permeability and oxidative phosphorylation) or 400 μM H2O2 that was added during last 4 hours of incubation. Plates were washed with PBS and then 100 μl of 10 μg/ml JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide, Invitrogen, T3168) added. Cells were incubated at 37 °C for 15 minutes in dark, washed twice with PBS, and 100 μl PBS was added to each well. Plates were read in SpectraMax M2 (Molecular Devices) to measure red J-aggregates fluorescence (a sensitive marker of Ψm) at an excitation of 535 nm and emission of 595 nm and green J-monomers fluorescence (indicator of disruption of Ψm) at an excitation of 485 nm and emission of 535 nm. Ratio of red/green fluorescence was calculated and plotted on the graph.
4
Mitochondrial Membrane Potential Assay in H9c2 Cells
5
Apoptosis and Mitochondrial Function Assays
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Cells were digested and rinsed with D-hank's then stained with Annexin V-FITC (5 μl diluted in 500 μl PBS)/PI (0.1 μl diluted in 500 μl PBS) apoptosis kit (CA1020, Solarbio, Beijing, Chian) according to the manufacturer's protocol. After, apoptotic cells were analyzed using flow cytometry (guava easyCyte™ 8, Millipore, USA). Mitochondrial ROS and mitochondrial membrane potential measurements were performed as published [12 (link)]. SH-SY5Y cells were stained with MitoSOX (2.5 μM, Invitrogen, USA) or with JC1 (10 μg/ml, T-3168, Invitrogen, USA) at 37 °C for 30 min. After washing with PBS twice, the cells were then resuspended in cold PBS containing 1% FBS for flow cytometric analyses. Data were analyzed with the FCS Express software (Guava Easy Cyte™8, Millipore, Hayward, CA, USA).
6
Mitochondrial Membrane Potential Assay
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Following a 6h co-treatment of FFA 0.75 mM and combinations of OLE and ROE, cells were incubated with a 3 μM JC-1 staining solution (T3168, Invitrogen, Waltham, MA, USA) at 37 °C for 20 min and then washed with PBS in order to visualize the fluorescence with a fluorescence microscope (EVOS Fl AMG). The JC-1 probe aggregates to form a polymer in the mitochondrial matrix of healthy cells, producing a strong red fluorescence (Ex = 585 nm, Em = 590 nm). Otherwise, dysfunctional mitochondria present JC-1 monomers, resulting in the emission of a green fluorescent signal (Ex = 514 nm, Em = 529 nm). The results are expressed as the ratio of red/green fluorescence.
7
Sperm Mitochondrial Membrane Potential Analysis
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The mitochondrial membrane potential of the sperm was evaluated by fluorescent multiple staining using propidium iodide (PI; 0.5 mg/mL), FITC-PNA (1 mg/mL in PBS), and 5,5’,6,6’-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1 (1.53 mM) diluted with DMSO (ratio 1:10) (T3168, Invitrogen, Waltham, MA, USA) staining. Then, 150 μL of frozen–thawed semen was mixed with 2.5 μL of PI and incubated at 37 °C for 5 min. Then, the mitochondria were labeled using 2 μL of JC-1 and incubated at 37 °C for 10 min. Finally, the acrosomes were labeled using 2 μL of FITC-PNA and incubated at 37 °C for 15 min in the dark [26 (link)]. Under a 400× magnification fluorescence microscope, 200 sperm were assessed. Midpiece staining showed that sperm with a high mitochondrial membrane potential fluoresced yellow-orange, whereas sperm with a low membrane potential fluoresced green (Figure 1 ). The percentage of sperm with high mitochondrial membrane potential was calculated [27 (link)].
8
Mitochondrial Membrane Potential Assay
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Following the treatments, cells were incubated with a 3 μM JC-1 staining solution (T3168, Invitrogen, Waltham, MA, USA) at 37 °C for 20 min and then washed with PBS in order to visualize the fluorescence with a fluorescence microscope (EVOS Fl AMG). The JC-1 probe aggregates to form a polymer in the mitochondrial matrix of healthy cells, producing a strong red fluorescence (Ex = 585 nm, Em = 590 nm). Otherwise, dysfunctional mitochondria present JC-1 monomers, resulting in the emission of a green fluorescent signal (Ex = 514 nm, Em = 529 nm). The results are expressed as the ratio of red/green fluorescence.
9
Mitochondrial Function and Senescence Analysis
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HKC‐8 cells were seeded on 6‐well plates and were given the specified treatments before staining. Then, cells were trypsinized, centrifuged at 600 g for 5 min, and then washed once with PBS. For JC‐1 staining and NAO staining, the cell pellets were suspended in 400 μl of JC‐1 (5 μM, T3168; Thermo Fisher) or NAO (5 μM, A1372; Thermo Fisher) solution, and were incubated at 37°C in the dark for 30 min. Then, cells were centrifuged, washed once, and resuspended in 400 μl of PBS. The cells were analyzed in a flow cytometry analyzer (BD FACSCalibur System). The relative mitochondrial membrane potential was calculated using the ratio of J‐aggregate/monomer (590/520 nm). Mitochondrial mass was measured by mean fluorescence intensity of NAO. For analysis of β‐gal‐positive cells, HKC‐8 cells were treated under the specified conditions, and then assessed using the FACS LacZ beta‐Galactosidase Intracellular Detection Kit (ab189816; Abcam) according to the manufacturer's protocol. Propidium iodide (PI) was used to exclude dead cells.
10
Mitochondrial Membrane Potential Assay by JC-1 Staining
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Mitochondria membrane potential was characterized by JC-1 staining (Thermo Fisher, T3168). Briefly, cells were seeded (2×104 per well) in Lab-Tek II chamber (NUNC™) 24 h prior to the treatment and cultured in a humidified atmosphere of 5 % CO2 and 95 % air at 37 °C. After 24 h incubation with CDN (with or without 10 μM of copper supplements) or control agents, cells were washed three times with PBS and incubated with JC-1 (working concentration of 10 μg/ml) in warm DPBS containing 10 μg/ml DAPI for 10 min. Then cells were washed three times with PBS and imaged with a confocal microscope (Zeiss LSM 710). For JC-1 monomers, 488 nm excitation wavelength was used and fluorescence signals at 525 ± 15 nm were collected as the green channel. For JC-1 aggregates, 546 nm excitation wavelength was used and fluorescence signals at 600 ± 15 nm were collected as the red channel.
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