Loss of mitochondrial membrane potential is indicative of apoptosis. ICT’s MitoPT TMRE Assay utilizes the potentiometric fluorescent dye TMRE to detect mitochondrial membrane permeability and membrane depolarization.
The TMRE dye has a delocalized positive charge dispersed throughout its molecular structure. In addition, its lipophilic solubility enables it to be readily membrane permeant and penetrate living cells. The TMRE dye enters the negatively charge mitochondria where it accumulates and fluoresces orange upon excitation. When the mitochondrial membrane potential collapses in apoptotic cells, MitoPT TMRE becomes distributed throughout the cytosol.
Detection of the loss of orange-red fluorescence in TMRE stained cells is a reliable method for assessing apoptosis induction or oxidative stress-induced mitochondrial depolarization in experimental cell populations.
1. Prepare samples.
2. Create controls with CCCP.
3. Dilute 10X Assay Buffer 1:10 with diH2O.
4. Reconstitute MitoPT TMRE with DMSO.
5. Dilute MitoPT TMRE with 1X Assay Buffer.
6. Add MitoPT TMRE to each sample.
7. Incubate 15-30 minutes.
8. Wash cells: add 1X Assay Buffer and spin cells.
9. Remove supernatant and resuspend cells for analysis.
10. Anazlyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. MitoPT TMRE excites at 549 nm and emits at 574 nm (orange).
Jurkat cells were cultured in the absence (healthy) or presence (dying) of staurosporine to induce apoptosis and then stained with ICT’s potentiometric dye TMRE. Cells were analyzed using a Nikon Eclipse E800 photomicroscope using a green excitation filter (510-560 nm) in tandem with a 570-620 nm emission filter. Apoptotic cells with depolarized mitochondria exhibit reduced orange/red fluorescence.
• Reagent name: TMRE
• Target: Mitochondrial depolarization
• Excitation/Emission: 549 nm / 574 nm
• Method of Analysis: Flow Cytometry, Fluorescence Microscope, Fluorescence Plate Reader
• Types of Samples: Cell culture
• Storage: ≤ -20°C
• Shipping: Ships overnight (domestic), International Priority Shipping
• MitoPT TMRE Reagent, 500 Tests, #6254
• 10X Assay Buffer, 2 x 125 mL, #6259
• CCCP, 50 mM, 600 µL, #6258
• Kit Manual
Rehklau K, Hoffmann L, Gurniak CB, Ott M, Witke W, Scorrano L, Culmsee C, Rust MB. Cofilin1-dependent actin dynamics control DRP1-mediated mitochondrial fission. Cell Death Dis. 2017. Oct 5;8(10):e3063. doi: 10.1038/cddis.2017.448. Abstract
” … overnight at 4 °C. Mitochondrial membrane potential. To quantify membrane potential, cells. were stained with the potentiometric fluorescent tetramethylrhodamine ethyl ester. (TMRE) dye (MitoPT TMRE kit; Immunochemistry Technologies, Bloomington, MN,. USA).