Description
Description: ICT's MitoPT TMRE Assay utilizes a well-characterized potentiometric fluorescent dye to detect mitochondrial membrane depolarization: tetramethylrhodamine ethyl ester, commonly known as TMRE. The lipophilic structure of TMRE allows it to easily penetrate cell and mitochondrial lipid bilayer membrane barriers. As a member of the rhodamine family of potentiometric dyes, TMRE will distribute itself within healthy, polarized mitochondria in a Nernstian manner. When accumulated in negatively charged, polarized mitochondria, TMRE emits a red fluorescence (em. 574 nm). When the mitochondrial membrane potential collapses in apoptotic or metabolically stressed cells, the MitoPT TMRE reagent 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 TMRE assay can be used in conjunction with existing apoptosis or metabolic stress protocols. Each kit includes the MitoPT TMRE potentiometric reagent, a wash buffer, and the mitochondrial membrane depolarizer Carbonylcyanide m- chlorophenylhydrazone (CCCP). TMRE-stained cells can be analyzed using flow cytometry, fluorescence microscopy, and fluorescence plate reader evaluation methodology.
Background: Detect Mitochondrial Permeability and Mitochondrial Membrane Depolarization with MitoPT ICT's MitoPT TMRE Assay utilizes the well-characterized potentiometric fluorescent dye tetramethylrhodamine ethyl ester to detect mitochondrial permeability and membrane depolarization. The rhodamine-based TMRE and TMRM dyes offered in ICT's MitoPT assays 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. Their potentiometric fluorescent properties allow for the detection of the mitochondrial permeability transition in whole cells via fluorescence analysis methods. MitoPT TMRE Assay Kit, 500 test size. Kit ASSAY PRINCIPLE: Rhodamine-based dyes like TMRE and TMRM are classified as slow potential sensitive dyes [1-2]. Compared to fast equilibration dyes that can redistribute in millisecond time intervals, slow potential sensitive dyes will require a few seconds to a minute to reach their equilibrium across an electrochemical gradient [1]. In addition to having a somewhat hydrophobic molecular structure, these membrane potential detecting redistribution dyes all have a delocalized positive charge dispersed across the fluorescent structure [2]. By doing so, they can more easily traverse the hydrophobic boundary layers of the cell and mitochondrial membrane structures [3]. Membrane potential driven accumulation of TMRE within the inner membrane region of healthy functioning mitochondria results in a dramatic increase in MRE-associated orange fluorescence. Experiments were performed to assess the respiratory cycle inhibitory affect of rhodamine 123, TMRM, and TMRE dyes during mitochondrial potential staining [4]. The data showed that all 3 rhodamine dyes demonstrated minimal toxicity when used at the nanomolar concentrations required to detect depolarization events. 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 [5]. Use of the MitoPT TMRE kit allows for the easy distinction between non-apoptotic cells and cells with depolarized mitochondrial membranes. Cells with polarized mitochondria will fluoresce red; cells with depolarized mitochondria will display diminished or no fluorescence. The MitoPT TMRE assay can be used in conjunction with existing apoptosis or metabolic stress protocols. Each kit includes the MitoPT- TMRE potentiometric reagent, a 10X wash buffer for removing any excess TMRE dye after staining, and the generic mitochondrial membrane depolarizer Carbonylcyanide m- chlorophenylhydrazone (CCCP). TMRE-stained cells can be analyzed using flow cytometry, fluorescence microscopy, and fluorescence plate reader evaluation methodology