JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry

TLDR

Mitochondrial membrane potential is a key indicator of cellular health, commonly assessed with the JC‑1 dye that shifts from green to red fluorescence in energized mitochondria and is usually excited at 488 nm in flow cytometry. Using 405 nm excitation on standard instruments yields cleaner JC‑1 aggregate signals with less monomer spillover, improving accuracy and removing the need for compensation, thereby benefiting mitochondria‑related research.

Abstract

Mitochondrial membrane potential provides a valuable indicator of cells’ health and functional status. Cytometry- and microscopy-based analyses, in combination with fluorescent probes, are widely used to study mitochondrial behavior related to cellular pathways, most notably – apoptosis. The cyanine dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi- dazolylcarbocyanine iodide) facilitates discrimination of energized and deenergized mitochondria because the normally green fluorescent dye forms red fluorescent aggregates when concentrated in energized mitochondria in response to their higher membrane potential. JC-1 fluorescence is usually excited by the 488 nm laser wavelength common in flow cytometers. In this study, we show that in practice this approach is not optimal for monitoring mitochondrial behavior. Investigation of fluorescence of JC-1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm excitation. The improved data are more accurate and eliminate the necessity for fluorescence compensation, making the use of the alternative excitation wavelengths beneficial for mitochondria-related biological and biomedial research.