Mitochondrial membrane potential (Δψ) was determined using a MitoPT TMRM assay kit according to manufacturer’s instructions (ImmunoChemistry Technologies, Bloomington, MN). Briefly, serum-deprived VSMCs were exposed to PIO (30 μM) for 1 or 3 hr, or vehicle control. Subsequently, VSMCs were stained with tetramethylrhodamine methyl ester (TMRM, 200 nM, 20 min at 37°C in the dark), washed with 1 ml wash buffer, and then visualized by fluorescence microscopy (Zeiss, Thornwood, NY). In intact cells, the cationic nature of TMRM allows it to accumulate within the inner membrane region of polarized mitochondria, resulting in a marked increase in TMRM-associated orange fluorescence. When mitochondrial membrane depolarizes, TMRM gets dispersed throughout the cytosol at a concentration that yields minimal fluorescence upon excitation. Thus, mitochondrial membrane potential was assessed by examination of TMRM-associated orange fluorescence (Excitation/Emission: 548 nm/573 nm). Carbonylcyanide m-chlorophenylhydrazone (CCCP, 50 μM, 1 hr) a generic mitochondrial membrane depolarizer, was used as a positive control.
Mitopt tmrm assay kit
Manufactured by ImmunoChemistry Technologies
Sourced in United States
The MitoPT TMRM assay kit is a laboratory tool that measures mitochondrial membrane potential. It utilizes the fluorescent dye tetramethylrhodamine methyl ester (TMRM) to detect changes in the electrochemical gradient across the mitochondrial membrane, a key indicator of mitochondrial function.
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3 protocols using mitopt tmrm assay kit
1
Measuring Mitochondrial Membrane Potential
2
Assessing PKR-Mediated Apoptosis in HeLa Cells
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HeLa cells were grown to 50% confluency in six-well plates and transfected with 500 ng of wt PKR pEGFPC1 and 20 ng pcDNA 3.1−, Flag TRBP AAAA/ pcDNA 3.1−, or Flag TRBP DDDD/pcDNA 3.1−. MitoPT® TMRM assay was performed using the manufacturer’s instructions (ImmunoChemistry Technologies MitoPT® TMRM Assay Kit). Green fluorescence (EGFP-PKR) and changes in red fluorescence (changes in mitochondrial polarization) were observed under an inverted fluorescence microscope (EVOS® FL Imaging System). At least 500 PKR expressing cells (GFP positive cells) were scored as live or dead based on decreased or absent red fluorescence. The percentage of cells undergoing apoptosis (Percent apoptosis) was calculated using the formula: (EGFP- expressing cells with decreased or absent red fluorescence/Total EGFP-expressing cells) × 100.
3
Mitochondrial Membrane Potential Assessment
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Mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester (TMRM) fluorescence imaging with a MitoPT TMRM Assay Kit (ImmunoChemistry Technologies, LLC, Bloomington, USA) according to the manufacturer's instruction. Fluorescence images were visualized by a confocal microscope (Olympus FV 1000, Olympus, Tokyo, Japan) at 543 nm excitation and 580 nm emission.
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