Loss of mitochondrial membrane potential is indicative of apoptosis. ICT’s MitoPT TMRM Assay utilizes the potentiometric fluorescent dye TMRM to detect mitochondrial membrane permeability and membrane depolarization.
The TMRM 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 TMRM dye enters the negatively charge mitochondria where it accumulates and fluoresces orange upon excitation. When the mitochondrial membrane potential collapses in apoptotic cells, MitoPT TMRM becomes distributed throughout the cytosol.
Detection of the loss of orange-red fluorescence in TMRM 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 TMRM with DMSO.
5. Dilute MitoPT TMRM with 1X Assay Buffer.
6. Add MitoPT TMRM 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. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. MitoPT TMRM excites at 548 nm and emits at 573 nm (orange).
Jurkat cells were cultured in the absence (healthy) or presence (dying) of staurosporine to induce apoptosis and stained with ICT’s potentiometric dye TMRM. Cells were analyzed using a Nikon 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: TMRM
• Target: Mitochondrial depolarization
• Excitation/Emission: 548 nm / 573 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 TMRM Reagent, 500 Tests, #6256
• 10X Assay Buffer, 2 x 125 mL, #6259
• CCCP, 50 mM, 600 µL, #6258
• Kit Manual
Wei J, Long L, Zheng W, Dhungana Y, Lim SA, Guy C, Wang Y, Wang YD, Qian C, Xu B, Kc A, Saravia J, Huang H, Yu J, Doench JG, Geiger TL, Chi H. Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy. Nature. 2019 Dec;576(7787):471-476. doi: 10.1038/s41586-019-1821-z. Epub 2019 Dec 11. Abstract
“For mitochondrial staining, lymphocytes were incubated for 30 min at 37 °C with 10 nM Mito Tracker Deep Red (Life Technologies) or 20 nM TMRM (ImmunoChemistry Technologies) after staining surface markers.”