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Product Detail

The MitoPT TMRM assay kits utilize a well-characterized, lipophilic but slightly cationic fluorescent dye to detect mitochondrial membrane depolarization: tetramethylrhodamine methyl ester, commonly known as TMRM. Both rhodamine-based dyes offered by ICT for this purpose (TMRE, TMRM), as well as the cationic JC-1 dye, are categorized as slow membrane redistribution dyes. The lipophilic structures of these dyes allow them to easily penetrate cell and mitochondrial lipid bilayer membrane barriers.

Use of the MitoPT TMRM kit allows for the easy distinction between healthy, non-apoptotic orange fluorescent cells and apoptotic or mitochondrial membrane-compromised minimally fluorescent cells. As a member of the rhodamine family of potentiometric dyes, TMRM will distribute itself within healthy, polarized mitochondria in a Nernstian manner. As this dye accumulates within the negatively charged mitochondrial organelles, the overall orange fluorescence (em. 573 nm) increases dramatically when excited at 548 nm. When the mitochondrial membrane potential collapses in apoptotic or metabolically stressed cells, the MitoPT TMRM reagent is no longer concentrated within the mitochondria. Instead, it is dispersed throughout the cell cytosol at a concentration that yields minimal fluorescence upon excitation in the optimal wavelength region (545-550 nm).

The MitoPT TMRM assay can be used in conjunction with existing apoptosis or metabolic stress protocols. Each kit includes the TMRM potentiometric dye reagent, a 10X wash buffer for removing any excess TMRM dye after staining, and the generic mitochondrial membrane depolarizer Carbonylcyanide m- chlorophenylhydrazone (CCCP). TMRM-stained cells can be analyzed using flow cytometry, fluorescence microscopy, and fluorescence plate reader evaluation methodology.