Answer: From a functional stand-point, JC-1, TMRM, and TMRE are all very similar. Healthy mitochondria have a charge gradient across the inner membrane; this is sometimes referred to as a transmembrane electrical potential. It is a proton and pH gradient and is normally negatively charged within the inner membrane space of healthy mitochondria. ICT’s mitochondrial depolarization detection dyes (TMRE, TMRM, and JC-1) are cationic, lipophilic dyes that enter healthy (polarized) mitochondria and accumulate, where they fluoresce orange upon excitation. When cells become apoptotic, stressed, or other pathologies arise, one of the first physiological indicators is a depolarization of the inner mitochondrial membrane. This means the dyes are no longer able to accumulate and instead are distributed more evenly across the cytosol. When this happens, overall orange cellular fluorescence levels drop dramatically. The purpose of ICT’s Mitochondrial Depolarization Assay Kits is to distinguish between healthy cells with polarized mitochondria and unhealthy cells with depolarized mitochondria.
Of our MitoPT products, TMRE (#9103) and TMRM (#9105) are closely related. Chemically they are very similar; TMRE is tetramethylrhodamine ethyl ester and TMRM is tetramethylrhodamine methyl ester. Peak excitation / emission of TMRE and TMRM is 549 / 574 nm and 548 / 573 nm, respectively. Of the two dyes, TMRE is brighter, having a greater fluorescence potential. TMRM exhibits the lowest mitochondrial binding and electron transport chain inhibition of any of these dyes, making it preferred for some studies. JC-1 (#924 and #911) is unique in that it exhibits a dual fluorescence property. In healthy cells with a high mitochondrial membrane potential, JC-1 spontaneously forms complexes known as J-aggregates with intense red fluorescence. On the other hand, in apoptotic, or unhealthy cells with low mitochondrial membrane potential, JC-1 remains in the monomeric form, which shows only green fluorescence. Flow cytometry analysis of JC-1 can be performed using 2 color red versus green fluorescence plots, or the ratio of green to red fluorescence allows comparative measurements of mitochondrial membrane potential between cell populations. The peak excitation of JC-1 is at 498 nm and the dual emission peaks (green and red) are at 525 nm and 595 nm, respectively. We should mention that all dyes are can be efficiently excited at 488 nm, despite this being slightly further from the peak for TMRE and TMRM.
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