Mitochondrial membrane potential was measured using TMRE Mitochondrial membrane Potential Assay Kit (Abcam), according to the manufacturer’s instructions. Fluorescence intensity was measured by flow cytometry (MACSQuant VYB) for >10,000 events in the final EPI gate were acquired for each sample. Each experiment was carried out in triplicate in at least two independent experiments. Fluorescence intensity was analyzed using FlowJo software (version 10.7.2).
Tmre mitochondrial membrane potential assay kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The TMRE-Mitochondrial Membrane Potential Assay Kit is a lab equipment product designed to measure the mitochondrial membrane potential in cells. It utilizes the cationic dye tetramethylrhodamine ethyl ester (TMRE) to stain mitochondria based on their membrane potential. The intensity of the fluorescent signal from TMRE is proportional to the mitochondrial membrane potential.
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Lab products found in correlation
Facscanto 2, by BD (3 mentions)
Lsm 880 laser scanning microscopy, by Bio-Rad (2 mentions)
Synergy htx microplate reader, by Agilent Technologies (2 mentions)
Zoe fluorescent cell imager, by Bio-Rad (2 mentions)
Synergy htx, by Agilent Technologies (2 mentions)
Facscalibur, by BD (2 mentions)
Software, by FlowJo (2 mentions)
Fcs express 7, by De Novo Software (2 mentions)
Fluorescence spectrophotometer, by Hitachi (2 mentions)
Complex 4 rodent enzyme activity microplate assay kit, by Abcam (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Mitosox red mitochondrial superoxide indicator kit, by Thermo Fisher Scientific (2 mentions)
Synergy ht multi detection microplate reader, by Agilent Technologies (1 mentions)
Atplite assay kit, by PerkinElmer (1 mentions)
Nad nadh glo assay kit, by Promega (1 mentions)
Ros glo assay kit, by Promega (1 mentions)
Glucose uptake glo assay kit, by Promega (1 mentions)
Lactate glo assay kit, by Promega (1 mentions)
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Cellview 4 compartment glass bottom tissue culture dishes, by Greiner (1 mentions)
82 protocols using tmre mitochondrial membrane potential assay kit
1
Measuring Mitochondrial Membrane Potential
2
Quantifying Mitochondrial Membrane Potential in HK2 Cells
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To quantify changes in mitochondrial membrane potential in HK2 cells, we used TMRE‐Mitochondrial Membrane Potential Assay Kit (Abcam catalogue ab113852) and measured fluorescence by microplate spectrophotometry. To label active mitochondria, we used TMRE (tetramethylrhodamine, ethyl ester, which is a cell permeant, positively charged, red‐orange dye that readily accumulates in active mitochondria due to their relative negative charge. Depolarized or inactive mitochondria have decreased membrane potential and fail to sequester TMRE. As a positive control, we used FCCP (carbonyl cyanide 4‐[trifluoromethoxy] phenylhydrazone), which is an ionophore uncoupler of oxidative phosphorylation. FCCP eliminates mitochondrial membrane potential and TMRE staining. We seeded HK2 cells (20 × 103) and allowed to attach for 24 h in 96‐well plates. We then replaced the culture media by fresh media with or without AAI at different concentrations for 24 h. FCCP was added to appropriate control cell samples at the end of experiment, and cells were incubated for 10 min. After washing cells with PBS (1X), we incubated them with TMRE for 15–30 min, washed again twice with PBS and then analysed using Bio‐tek Synergy™ HT Multi‐Detection Microplate Reader (Biotek®, Winooski, VT, USA) at Ex/Em 549/575 nm.
3
Metabolic Profiling of Cell Cultures
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MMP was investigated using the TMRE Mitochondrial Membrane Potential Assay Kit (Abcam). Intracellular ATP level at steady state was measured using ATPlite Assay Kit (PerkinElmer). Lactate level was measured using Lactate-Glo Assay Kit (Promega). Glucose uptake was assessed using Glucose Uptake-Glo Assay Kit (Promega). NAD+ and NADH levels were measured using NAD/NADH-Glo Assay Kit (Promega). ROS level was measured using ROS-Glo Assay Kit (Promega). The assays were performed according to the manufacturer's instructions. See supplemental information for detailed procedures.
4
Mitochondrial Dysfunction Evaluation in HUVECs
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Mitochondrial dysfunction was evaluated based on mitochondrial membrane potential. This was measured using a tetramethylrhodamine, ethyl ester (TMRE)-Mitochondrial Membrane Potential Assay Kit (Ex/Em = 549/575 nm; Abcam). After HUVECs were seeded in 6-well plates (2 × 104 cells/well), the cells were treated with TGF-β2 and IL-1β, as well as 0, 1, 5, 10 μM EGCG, and incubated for 2 h at 37 °C. Then, the medium was changed to medium containing 100 nM TMRE and incubated for 30 min at 37 °C without light.
5
Mitochondrial Membrane Potential Imaging
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To access the changes in mitochondrial membrane potential, tetramethylrhodamine ethyl ester (TMRE) Mitochondrial Membrane Potential Assay Kit (Abcam, ab113852) was utilized as per manufacture’s protocol. Briefly, Cells were plated in CELLview 4‐compartment glass‐bottom tissue culture dishes (Greiner Bio‐One, 627,870), and treated as per experimental condition. Cells were then incubated for 30 min at room temperature with 50 nM TMRE and and NucBlue™ Live ReadyProbes™ Reagent (Thermo Fisher Scientific, R3760) and proceed for live‐cell imaging with the alpha Plan‐Apochromat 100×/1.46 Oil DIC M27 objective on the Zeiss LSM 880 with Airyscan. Prior to image analysis, raw .czi files were automatically processed into deconvoluted Airyscan images using the Zen software. Leica SP8 LSM, fitted with STED module, was used to perform live‐cell super‐resolution imaging.
6
Apoptosis and Mitochondrial Analysis
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An Annexin V-FITC Kit (Beyotime, C1062) was used to detect the apoptotic cells. For determining mitochondrial membrane potential (MMP) and analyzing ROS production, cells were stained using the TMRE Mitochondrial Membrane Potential Assay Kit (Abcam, Cambridge, UK, ab113852) and MitoTracker® Red CMXRos (Yeasen, 40741ES50) and analyzed by flow cytometry (FACSCanto, BD) within 1 h.
7
Quantifying Mitochondrial Membrane Potential in NSCLC
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A TMRE-Mitochondrial Membrane Potential Assay Kit (ab113852, Abcam) was used to quantify changes in mitochondrial membrane potential in live NSCLC cells, according to the manufacturer’s instructions. Briefly, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 5 μM) was added into active NSCLC cell suspensions, including appropriate control cell samples, and incubated for 10 min at 37 °C, 5% CO2. Tetramethylrhodamine ethyl ester (TMRE, 200 nM) was then added to the cell suspensions and incubated for another 30 min. Suspended cells were pelleted by 200× g for 5 min and washed with PBS buffer supplemented with 0.2% BSA. The fluorescence was detected by flow cytometry (LSRII) through the channel of 488 nm/575 nm (excitation/emission).
8
Mitochondrial Membrane Potential Assay
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The changes in mitochondrial membrane potential were determined using a tetramethylrhodamine ethyl ester perchlorate (TMRE) mitochondrial membrane potential assay kit (Abcam) following the manufacturer’s instruction. The AML-12 cells were seeded into 96-well plates at a cell density of 1 × 104 cells per well and cultured for 24 h. These cells were treated with a medium containing recombinant TGF-β1 at 5 ng/mL and DIM at indicated concentrations (0, 10, 20, and 40 µM) for 24 h. The medium was then discarded, and the cells were stained with TMRE (400 nmol/L) for 20 min at room temperature in the dark. After the cells were washed twice with PBS, TMRE intensity was measured at excitation and emission wavelengths of 549 nm and 575 nm, respectively, using a fluorescence plate reader.
9
Mitochondrial Membrane Potential Assay
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For mitochondrial membrane potential measurements, a TMRE-Mitochondrial Membrane Potential Assay Kit (abcam, ab113852) was used according to manufacturer’s instruction. Briefly, hESC-RPE cells were thawed on a 48 well plate and cultured for 3 and 12 weeks, respectively. 600 nM tetramethylrhodamine ethyl ester (TMRE) solution diluted in culture media was used, and 20 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) served as a negative control compound. All steps were carried under light protection. Diluted TMRE was added to the cells and incubated for 20 min at + 37 °C, and a microplate reader was used to detect the fluorescence from the TMRE. Excitation and emission wavelengths of 549/575 nm were used. Negative controls were measured for both treated and non-treated cells. Results from the microplate reader were given as intensity values and the values are presented as boxplots. Control values were set to 100% and values from the treated cells were compared to the control cells as relative change in the fluorescence intensity. Higher fluorescence intensity corresponds to a higher value of mitochondrial membrane potential. 13–16 replicate wells from 3 maturation experiments were measured at both time points.
10
TMRE Assay for Mitochondrial Membrane Potential
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Active mitochondria were measured with TMRE Mitochondrial Membrane Potential Assay kit (Abcam, Cambridge, UK). U33 cells were seeded in 24‐well plates at a density of 25,000 cells/well and allowed to grow for 24 hours. Cells were treated with 100 ng/mL of either rCPE or mrCPE protein for 24 hours, followed by a change of media supplemented with 40nM TMRE (tetramethylrhodamine ethyl ester perchlorate). Cells were incubated for 20 min, media were aspirated, and replaced with 100‐mL PBS/0.2% BSA (twice), according to the manufacturer's protocol. The fluorescent intensity of TMRE accumulated in active mitochondria was measured using the Cytation 5 plate reader (BioTek Instruments) at excitation/emission wavelengths of 549/575 nm.
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