EndoC-βH1 cells were seeded onto collagen-coated 8-well chambered cover glasses (Lab-Tek, Thermo Scientific) at a density of 70,000 cells/cm2. After 24 h, cells were transfected with 100 nM of DIMT1 siRNA and incubated further for 72 h. For the ΔΨm measurement, cells were preincubated with imaging buffer (135 mM NaCl, 3.6 mM KCl, 1.5 mM CaCl2, 0.5 mM MgSO4, 0.5 mM Na2HPO4, 10 mM HEPES, 5 mM NaHCO3, pH 7.4) containing 1 mM glucose for 2 h with 100 nM of tetramethylrhodamine methyl ester (TMRM; Invitrogen). This imaging buffer formulation was standardized for the microscopy studies. After the incubation, cells were washed again with the imaging buffer only and subjected to live-cell confocal microscopy in quench mode, where the whole-cell fluorescence decreases upon mitochondrial hyperpolarization. After recording the basal level of TMRM fluorescence in 1 mM glucose, cells were switched to 20 mM glucose. Carbonyl cyanide-4-phenylhydrazone (FCCP) was used to dissipate the ΔΨm. Zeiss LSM510 inverted confocal fluorescence microscope with 543 nm excitation and 585 nm long pass emission settings were used to record the data, which were background corrected and normalized.
Tetramethylrhodamine methyl ester tmrm
Manufactured by Thermo Fisher Scientific
Sourced in United States, France, Ireland
Tetramethylrhodamine methyl ester (TMRM) is a fluorescent dye used in biological research. It is a cell-permeant cationic dye that accumulates in active mitochondria due to their large membrane potential. TMRM can be used to monitor mitochondrial membrane potential and assess mitochondrial function in living cells.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
Rotenone, by Merck Group (6 mentions)
Mitosox red, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
β actin, by Cell Signaling Technology (3 mentions)
D glucose, by Merck Group (3 mentions)
Thiazolyl blue tetrazolium bromide, by Merck Group (3 mentions)
Oligomycin, by Merck Group (3 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (3 mentions)
Mitosox, by Thermo Fisher Scientific (3 mentions)
Mitotracker green, by Thermo Fisher Scientific (3 mentions)
Facscalibur, by BD (3 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
2 deoxy d glucose, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
62 protocols using tetramethylrhodamine methyl ester tmrm
1
Evaluation of Mitochondrial Membrane Potential
2
Mitochondrial Dynamics Regulation in Toxicity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isoniazid and SB203580 (SB) were obtained from Sigma-Aldrich (St. Louis, MO, United States). Mdivi-1, an inhibitor of DRP1, was purchased from Selleck Chemicals (Houston, TX, United States). Tetramethyl rhodamine methyl ester (TMRM), MitoTracker Deep Red FM, Hoechst 33342 and Lipofectamine RNAiMAX were obtained from Invitrogen (Grand Island, NY, United States). p38 MAPK-siRNA, a silencer negative control siRNA, and antibodies against p38 MAPK, phospho-p38 MAPK, NRF1, COX IV, cytochrome c, caspase 9, caspase 3, MFN2, DRP1, acetylated lysine, p-MAPKAPK-2, MAPKAPK-2 and β-actin were purchased from Cell Signaling Technology (Danvers, MA, United States). Antibodies against SIRT1 and Bax were obtained from Abcam (Abcam, Cambridge, United Kingdom). PGC1α antibody and protein A/G-agarose beads were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). All other chemicals were of analytical grade.
3
Cell Metabolic Profiling Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were of the highest purity available. The fluorescence dyes, tetramethylrhodamine methyl ester (TMRM) and Hoechst 33342, were from Invitrogen (Carlsbad, CA). Cell culture media and supplements were from Invitrogen (Carlsbad, CA). Low-buffered RPMI medium was from Molecular Devices (Sunnyvale, CA). XF96 sensor cartridges and XF96-well plates were from Seahorse Bioscience (Billerica, MA).
4
Multiparametric Evaluation of Cell Viability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was determined by propidium iodide (PI) dye exclusion. After treatment, cells were incubated with 10 µg/ml PI (Sigma‐Aldrich, Dorset, UK) and the integrity of cell membrane was measured by flow cytometry using a FACS Canto (Becton Dickinson, Oxford, UK). To determine the mitochondrial membrane potential (ΔΨm), cells were stained with 40 nM tetramethylrhodamine methyl ester (TMRM; Invitrogen) at 37°C for 15 min and changes in ΔΨm were measured on the FH2‐H channel (Liu, Agarwal, Movasaghi, et al., 2008 ). To measure the production of reactive oxidative species (ROS), cells were stained with 40 µM dihydroethidium (HE; Polyscience‐Park Scientific, Northampton, UK) at 37°C for 15 min and increases in ROS production were measured on the FH3‐H channel (Liu, Agarwal, Movasaghi, et al., 2008 ). For mitochondrial cytochrome c release, cells were permeabilized or fixed as previously reported (Liu et al., 2010 ) and washed three times with phosphate buffer saline (PBS). Cells were incubated with 20 µl of PE‐conjugated anti‐cytochrome c mAb (Santa Cruz Biotechnology, Inc., Dallas, TX) for 1 hr at room temperature in the dark. Before analysis by flow cytometry, cells were washed once in PBS.
5
Mitochondrial Membrane Potential Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondria are the key subcellular organelles for many anti-cancer drugs to induce apoptosis and the loss of mitochondrial membrane potential is a major factor in the mitochondrial induced apoptosis pathway.23 To detect whether the mitochondrial pathway is involved in apoptosis induced by 5-acetamido-1-(methoxybenzyl) isatin, K562 cells (5 × 104 cells per mL) were seeded in six-well plates and allowed to grow 2 h, then treated with DMSO or 5-acetamido-1-(methoxybenzyl) isatin (1 μM) for 0, 6, 12, 24 and 48 h respectively. Washed cells with PBS and stained with 100 nM tetramethylrhodamine methyl ester (TMRM; Invitrogen, USA), then left it in the dark for 15 min at room temperature. At the end, distinction of mitochondrial membrane potential was analyzed by flow cytometry in the FL-2 channel.
6
Mitochondrial Membrane Potential Assay
7
Mitochondrial Membrane Potential Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial membrane potential (ΔΨm) serves as a sensitive measure of mitochondrial activity and physiological status. NGC0211 cells (1 × 104 cells/well/100 µL) were plated onto clear bottom 96-well black plates (Corning, New York, NY, USA) and allowed to grow for 24 h. Cells were then treated with the control medium or MCMs for 3 or 24 h, respectively. At 30 min prior to treatment completion, 0.2 µM tetramethyl rhodamine methyl ester (TMRM, final concentration) (Invitrogen, Waltham, MA, USA) was added. Cells were then washed twice with 100 µL phosphate-buffered saline (PBS) (Invitrogen, Waltham, MA, USA), and the fluorescence at 548/574 nm was read using a spectrophotometer (Safire II Plate reader, Tecan, Männedorf, Switzerland; 5 nm bandpass; bottom read).
8
Cytotoxicity Evaluation of Venetoclax and WEHI-539
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fetal bovine serum, RPMI 1640 medium, Thiazolyl Blue Tetrazolium Bromide (MTT), dimethyl sulfoxide (DMSO), sodium pyruvate, D-glucose, 2-deoxy-D-glucose, Hoechst 33588 and propidium iodide (PI) came from Sigma-Aldrich (Dublin, Ireland). DMEM medium was purchased from Lonza (Analab Ltd, Lisburn, United Kindom). Tetramethylrhodamine methyl ester (TMRM) was from Invitrogen (Biosciences, Ireland). Venetoclax was purchased from Active Biochem (Maplewood, NJ, USA), WEHI-539 from ChemScene (South Brunswick, NJ, USA).
9
Lipid and Metabolite Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS) and Trypsin were purchased from Gibco-Invitrogen (Grand Island, NY, USA). Dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF), protease inhibitor cocktail (leupeptin, antipain, chymostatin, and pepstatin A), sulforhodamine B (SRB), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), arachidonic acid (C20:4), D(-)-Fructose, Nile Red, Hoechst 33342, tetramethylrhodamine methyl ester (TMRM), and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) were purchased from Invitrogen (Eugene, OR, USA).
10
Mitochondrial Dynamics Regulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Small molecule S-3 was gifted by Professor Quan Chen37 (link). D-glucose, sorbitol, menadione, mitoTEMPO, cyclosporine A (CsA), and rotenone were from Sigma. Tetra-methylrhodamine methyl ester (TMRM) was from Invitrogen. SS31 (D-Arg-Dmt-Lys-Phe-NH2) was synthesized as described44 (link). Antibody against Drp1 was obtained from BD Biosciences.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!