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

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.

A comparison of radiation-induced mitochondrial damage between neural progenitor stem cells and differentiated cells

“…The mitochondrial membrane potential (DCm) in NSCs and NCs was measured by staining with the lipophilic cation JC-1. 23 As expected, long-term FR elevated the DCm in NCs (Fig. 3A, right panel).…”

Synthesis and evaluation of selected 1,3,4-oxadiazole derivatives for in vitro cytotoxicity and in vivo anti-tumor activity

“…A polarized mitochondrial membrane draws additional dye into the mitochondrial membrane from the medium and reduces the fluorescence intensity of the suspension. Whereas if there is depolarization or any damage to the mitochondrial membrane potential dyes is released dye into the medium, increasing the overall fluorescence (Perelman et al 2012). Our results confirmed the anticancer potential of the tested compounds on Hep2 cells by increasing the fluorescence intensity, which was a direct confirmation of the mitochondrial damage and change in the mitochondrial membrane potential as the possible mechanism for the anticancer property of the tested compounds.…”

Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

“…47 Briefly, JC-1 was added to reach a final concentration of 5 mg/ml and incubated for 20 min, and then the cells were washed twice with medium and imaged under a fluorescence microscope.…”