Detect loss of mitochondrial membrane potential using ICT's innovative system with the MitoPT reagent. In non-apoptotic cells with intact mitochondrial membrane potential, the MitoPT reagent aggregates in mitochondrial cells and fluoresces red (590 nm emission). When cell undergo apoptosis and loose potential across the mitochondrial membrane, the MitoPT dye is dispersed throughout the cell in a monomeric form that fluoresces green (527 nm emission). Thus, healthy cells fluoresce red and apoptotic cells fluoresce green-making them very easy to differentiate.

Detection of the mitochondrial permeability transition event (PT) provides an early indication of the initiation of cellular apoptosis. This process is typically defined as a collapse in the electrochemical gradient across the mitochondrial membrane, as measured by the change in the membrane potential (DY).

Loss of mitochondrial DY, indicative of apoptosis, can be detected by a unique fluorescent cationic dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzamidazolocarbo-
cyanin iodide, commonly known as JC-1¹. This dye has been incorporated into the user-friendly MitoPT kit for the simple and reproducible detection of the PT event in apoptotic cells.

In Figures 4 and 5, cells were analyzed in a Becton Dickinson FACSCaliber Flow Cytometer. Jurkat cells were treated with either DMSO (negative, non-induced cells) or with staurosporine (apoptotic, induced cells) for 3 hours at 377C, then labeled with MitoPTT for 15 minutes. Collapse of the mitochondrial DY is indicated by an increase in the number of cells falling into R3 corresponding to a loss of red fluorescence, indicative of the onset of apoptosis. (In these figures of flow cytometer data, non-apoptotic cells are in R2, which prints in green, and apoptotic cells are in R3, which prints in pink.)

Reference:

1. Smiley, S. T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T. W., Steele, G.D., and Chen, L. B. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA 88: 3671-3675 (1991).