Background

Apoptosis is a cellular process which involves genetical events causing the death of a cell. Several major events occur in mitochondria, of which, the most significant is the loss of mitochondrial transmembrane potential (ΔΨ_M) (Green and Reed, 1998). Throughout the life of a cell, the mitochondria uses oxidizable substrates to produce an electrochemical proton gradient across the mitochondrial membrane which is used to produce ATP. The direction of the mitochondrial membrane potential (with the interior of the organelle being electronegative) is such to produce inward transport of cations and outward transport of anions, thus promoting accumulation of cations in the mitochondria (Zorova et al., 2018). This electrochemical gradient drives the synthesis of ATP. However, during apoptosis, ΔΨ_M decreases as the process is associated with the opening of the mitochondrial permeability pores and loss of the electrochemical gradient. Thus, ΔΨ_M is an essential parameter of the mitochondrial function that can be used as an indicator of cell health since mitochondria are inherently involved in the apoptotic process of cells.

In recent years, ΔΨ_M has been studied using several fluorescent cationic dyes including rhodamine-123 and DiOC₆ as common monitoring tools (Cossarizza and Salvioli, 1998). The technique involving using 5,5,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1) dye has been developed to detect ΔΨ_M in healthy and apoptotic cells across multiple cell types, such as neurons, myocytes, endothelial cells etc. JC-1 can be used to assess the ΔΨ_M both in intact isolated mitochondria and tissues. In fact, the JC-1 dye is a lipophilic, cationic dye (naturally exhibiting green fluorescence) which is able to enter into the mitochondria where it accumulates and (in a concentration-dependent manner) starts forming reversible complexes called J aggregates. Differently, from JC-1 molecules, these J aggregates exhibit excitation and emission in the red spectrum (maximum at ~590 nm) instead of green. Thus, in healthy cells with a normal ΔΨ_M, the JC-1 dye enters and accumulates in the energized and negatively charged mitochondria and spontaneously forms red fluorescent J-aggregates. By contrast, in unhealthy or apoptotic cells the JC-1 dye also enters the mitochondria but to a lesser degree since the inside of the mitochondria is less negative because of increased membrane permeability and consequent loss of electrochemical potential. Under this condition, JC-1 does not reach a sufficient concentration to trigger the formation of J aggregates thus retaining its original green fluorescence.

Based on these premises, the red/green fluorescence ratio of the dye in the mitochondria can be considered as a direct assessment of the state of the mitochondria polarization whereas the higher is the ΔΨ_M, the more elevated is the red shift of the dye (more J aggregates are formed). Vice-versa; the lower is the ΔΨ_M of the mitochondria and the lower is the red to green ratio of the fluorescent marker (few J aggregates are formed). Therefore, mitochondrial depolarization is indicated by a reduction in the red to green fluorescence intensity ratio.

The aggregate green monomeric form has absorption/emission of 510/527 nm, and the aggregate red form has absorption/emission 585/590 nm (Smiley et al., 1991). The red to green fluorescence intensity ratio is only dependent on the membrane potential and no other factors such as shape, mitochondrial size, and density, which may influence single-component fluorescence signals. It is noteworthy JC-1 dye can be used both as qualitative (considering the shift from green to red fluorescence emission) and quantitative (considering only the pure fluorescence intensity) measure of mitochondrial membrane potential (Cossarizza and Salvioli, 1998).

Fluorescent dye accumulation in mitochondria can be optically detected by flow cytometry, fluorescent microscopy, confocal microscopy, and through the use of a fluorescence plate reader (Perry et al., 2011). Indeed, the use of fluorescence ratio detection provides researchers with a means to compare measurements of membrane potential while also assessing the percentage of mitochondrial depolarization occurring in a pathological condition (e.g., cellular stress, apoptosis, etc.).

Thus, the main objective of this paper is to provide a practical step by step user protocol to assess and monitor mitochondrial membrane potential in whole cells using the JC-1 fluorescent cationic probe. As a positive control, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) which is a chemical inhibitor of oxidative phosphorylation, affecting the protein synthesis reactions in seedling mitochondria and causing the gradual destruction of living cells and death of the organism is used. In order to illustrate an example for the use of the JC-1 dye, we also provide data analysis related to the mitochondrial membrane potential of two types of mouse-derived brain microvascular endothelial cells subjected to oxidative stress insults by exposure to tobacco.