The mitochondrial membrane potential of cells treated with b-AP15 or untreated were assayed by mitochondrial membrane potential kit (Sigma-Aldrich, St. Louis, MO), following manufacturer’s instruction. DLBCL cells were treated with various doses of b-AP15, and after 24 h the cells were harvested, prepared in 1 ml warm medium, and then 5 μl cationic hydrophobic mitochondrial potential dye was added. The cells were incubated for 30 min in a 5% CO2, 37 °C incubator. After centrifugation the cells were resuspended with 500 μl assay buffer, followed by monitoring the cells using a flow cytometry with ƛex = 635 nm, ƛem = 660 nm at APC channel.
Mitochondrial membrane potential kit
Manufactured by Merck Group
Sourced in United States
The Mitochondrial Membrane Potential Kit is a laboratory tool used to measure the electrochemical gradient across the mitochondrial membrane, which is a critical indicator of cellular energy production and mitochondrial function.
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Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp, by Merck Group (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
96 well plate, by Corning (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Zno np, by Merck Group (1 mentions)
Antibiotics antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Anti pink1 ab23707, by Abcam (1 mentions)
Anti vdac1, by Cell Signaling Technology (1 mentions)
Anti lc3b, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti caspase 9, by Cell Signaling Technology (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Dna ladder, by HiMedia (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
18 protocols using mitochondrial membrane potential kit
1
Mitochondrial Membrane Potential Assay
2
Mitochondrial Membrane Potential Assay
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The mitochondrial membrane potential (ΔΨm) was measured using JC-10 dye for determining the loss of the MMP upon PA treatment. In brief, HepG2 cells were seeded in a 96-well plate and treated with 0.5 mM concentrations of PA for 24 h, and subjected to staining according to the manufacturer’s instructions (Sigma-Aldrich, Mitochondrial Membrane Potential Kit. (Cat no. MAK159)). JC-10 was formed fluorescent aggregates according to the polarization of the mitochondrial membrane upon PA treatment. Fluorescence intensity (lex = 490/ lem = 525 nm) and (lex = 540/lem = 590 nm) were monitored for ratio analysis using ELISA plate reader (SpectraMax M5, Molecular Devices). The ratio of red/green fluorescence intensity was calculated using GraphPad Prism 7.00 software.
3
Mitochondrial Membrane Potential Assay in Candida
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The change in the mitochondrial membrane potential (MMP) of C. albicans cells was analyzed using JC-10 (analog of JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide). For this purpose, the Mitochondrial Membrane Potential Kit (Sigma-Aldrich, USA) was used. The cells treated with quinalizarin at 2, 4, and 16 µg/mL for 5 h were washed and stained with JC-10 for 30 min in the dark. The red fluorescence intensity (λex = 540 nm/λem = 590 nm) and green fluorescence intensity (λex = 490 nm/λem = 525 nm) were monitored using a microplate reader. The ratio of red/green fluorescence intensity was used to determine MMP. An untreated sample and a 50 µM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)-treated (Sigma-Aldrich, USA) sample were used as negative and positive controls, respectively.
4
Assessment of Mitochondrial Functionality
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Tail tip fibroblasts were seeded in 96-well plates and the mitochondrial membrane potential (MMP) determined using the mitochondrial membrane potential kit (Sigma) following the manufacturer’s recommendations. The red/green fluorescence intensity ratio was used to determine MMP values. To determine mitochondrial activity, fibroblasts were stained with 4 nM tetramethylrodamine for 30 min in complete RPMI medium without phenol red at 37°C and 5% CO2. Samples were analyzed using an Olympus FV1000 confocal microscope and FV10 software (Olympus). Additional determination of MMP was done using the JC-10 dye. Fibroblasts were processed as indicated above and incubated with JC-10 for 30 min at 37°C and 5% CO2. Samples were analyzed using a Cytoflex S flow cytometer (Beckman Coulter).
5
Mitochondrial Membrane Potential Assay
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ΔΨm staining was performed using Mitochondrial Membrane Potential Kit (Sigma Aldrich, United States), 5000 ARPE-19 cells/well were seeded in 96-well plates (Corning, United States) and treated with Arbutin and TBHP for certain hours, and 100 μL of JC-1 solution was added to the cells for 20 min. The absorbance at 514/529 and 585/590 nm were detected using a microplate reader (BioTek Instruments, Winooski, Vermont, United States), and cells were observed under a fluorescent inverted microscope.
6
Apoptosis Assay for IEC Monolayers
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Moreover, a JC-10 assay was conducted to test for the apoptosis of the IEC monolayers 48 h after treatment and stimulation. The Mitochondrial Membrane Potential Kit (Sigma Aldrich, St. Louis, MO, USA) was utilized in accordance with the manufacturer’s instructions. Valinomycin (val, 100 µg/mL) served as a positive control for apoptosis.
7
Investigating Autophagy and Oxidative Stress
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ZnO NPs, dansylcadaverine (MDC), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and the Mitochondrial Membrane Potential Kit were purchased from Sigma Chemical (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium and α-MEM culture media, the antibiotics–antimycotic solution, and fetal bovine serum were purchased from GIBCO (Grand Island, NY, USA). Anti-LC3B, anti-VDAC1, anti-p53, anti-caspase 9, and anti-GAPDH antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-PINK1 (ab23707) and anti-parkin (ab77924) were purchased from Abcam (Cambridge, MA, USA). MitoTracker Red CMXRos and Lipofectamine 2000 (11668-027) were purchased from Invitrogen (Carlsbad, CA, USA). GFP-LC3 was supplied by Prof Tanfeng from Wenzhou Medical University.
8
Mitochondrial Membrane Potential in PC12 Cells
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PC12 cells were plated at a density of 8 × 104 cells per well in 96-well black plate and incubated for 24 h at 37 ± 2 °C in a 5% CO2-humidified incubator. Supernatant was removed and pre-treated with fresh medium containing 0.25 mg/mL P. australis ethanol extract or 10 µg/mL desipramine for 2 h before exposure to 600 µM corticosterone for 24 h. Supernatant was then subjected to MMP measurement according to the manufacturer’s protocol of mitochondrial membrane potential kit (Sigma-Aldrich, St. Louis, MO). Fluorescence intensity shift from red to green was determined by measuring the absorbance at an excitation and emission wavelength of 540 nm/590 nm (red) and 490 nm/525 nm (green) in a UV–Vis spectrophotometer microplate reader and expressed as a percentage relative to negative control.
9
Evaluation of Anticancer and Antioxidant Properties
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Bovine serum albumin (BSA), nitro blue tetrazolium
(NBT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), 2,2-diphenyl-1-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO),
and DNA ladder were purchased from HiMedia. 2′,7′-Dichlorofluorescein
diacetate (DCFH-DA), TRIzol reagent, and the mitochondrial membrane
potential kit were purchased from Sigma. The cDNA synthesis kit was
purchased from verso, the PCR master mix was purchased from Genei,
and primers were purchased from Eurofins. Human cancer cell lines
A549, HepG2, and MCF-7 were procured from NCCS Pune. All other chemicals
used were of analytical grade procured from reputed firms.
(NBT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), 2,2-diphenyl-1-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO),
and DNA ladder were purchased from HiMedia. 2′,7′-Dichlorofluorescein
diacetate (DCFH-DA), TRIzol reagent, and the mitochondrial membrane
potential kit were purchased from Sigma. The cDNA synthesis kit was
purchased from verso, the PCR master mix was purchased from Genei,
and primers were purchased from Eurofins. Human cancer cell lines
A549, HepG2, and MCF-7 were procured from NCCS Pune. All other chemicals
used were of analytical grade procured from reputed firms.
10
Cytotoxicity and Complement Inhibition Assay
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Sodium tripolyphosphate (TPP) was purchased from Acros Organics and sterile water from Laboratoire Aguettant, Lyon, France. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), chitosan (CS, low viscosity from shrimp shells), sodium hyaluronate, sodium pyruvate, penicillin-streptomycin-amphotericin antibiotic mixture, hemoglobin from bovine blood, Triton X-100, mitochondrial membrane potential kit, and In Vitro Toxicology Assay Kit Lactic Dehydrogenase based were acquired from Sigma–Aldrich (St. Louis, MO, USA). Cell culture reagents and culture medium were provided by Gibco (Grand Island, NY, USA). The RAW 264.7 cell line was obtained from American Type Culture Collection (ATCC-TIB 71), Rockville, MD, USA. Complement MicroVue CH50 Eq Enzyme Immunoassay Kit was purchased from Quidel (Quidel, San Diego, CA, USA).
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