JC-10 probe as a novel method for analyzing the mitochondrial membrane potential and cell stress in whole zebrafish embryos

Abstract

Background A sensitive method to investigate cellular stress and cytotoxicity is based on measuring mitochondrial membrane potential. Recently, JC-10, was developed to measure mitochondrial membrane potential in vitro and used as an indicator for cytotoxicity. Yet, JC-10 has never been used in vivo (whole organism). In normal cells, JC-10 concentrates in the mitochondrial matrix, where it forms red fluorescent aggregates. However, in apoptotic/necrotic cells, JC-10 diffuses out of the mitochondria, changes to monomeric form, and stains cells in green. Here, we aimed to develop and optimize a JC-10 assay to measure cytotoxicity in zebrafish embryo. We also investigated the effectiveness of JC-10 assay by comparing it to common cytotoxicity assays. Methods Zebrafish embryos were exposed to a toxic surfactant AEO-7 at no observed effect concentration (6.4 μg/L), and then cytotoxicity was measured using (i) JC-10 mitochondrial assay, (ii) acridine orange (AO), (iii) TUNEL assay, and (iv) measuring the level of Hsp70 by western blotting. Results As compared to the negative control, embryos treated with NOEC of AEO-7 did not show significant cytotoxicity when assessed by AO, TUNEL or western blotting. However, when JC-10 was used under the same experimental conditions, a significant increase of green:red fluorescent ratio signal was detected in the AEO-7 treated embryos, indicating mitochondrial damage and cellular cytotoxicity. Noteworthy, the observed green: red ratio increase was dose dependent, suggesting specificity of the JC-10 assay. Conclusion JC-10 is a sensitive in vivo method, thus, can be used as surrogate assay to measure cytotoxicity in whole zebrafish embryos.

Carboxymethyl Chitosan-Coated Cyanidin-3-O-Glucoside-Beared Nanonutriosomes Suppress Palmitic Acid-Induced Hepatocytes Injury

“…Additionally, we assessed the collapse of mitochondrial membrane potential (MMP) using JC-10 fluorescence probe. 48 Figure 6C,D illustrated that 0.15 mM of PA treatment greatly declined the ratio of red to green fluorescence, suggesting that PA exposure reduced the MMP compared to the control group. On the contrary, treatment of free-C3G, C3G-NS, and CMC-C3G-NS significantly improved the MMP by 69.88 ± 1.12%, 85.80 ± 0.68%, and 87.90 ± 1.91%, respectively.…”

“…Additionally, we assessed the collapse of mitochondrial membrane potential (MMP) using JC-10 fluorescence probe Figure C,D illustrated that 0.15 mM of PA treatment greatly declined the ratio of red to green fluorescence, suggesting that PA exposure reduced the MMP compared to the control group.…”

Antifungal activity of human antimicrobial peptides targeting apoptosis in Candida auris

“…In healthy cells, this dye forms red fluorescent aggregates, while in apoptotic cells with low mitochondrial membrane potential, it spreads across the cytoplasm in green fluorescent monomers. The magnitude of mitochondrial membrane potential was assessed by determining the JC-10 aggregates to monomers ratio, the increase in JC-10 monomers associated with apoptosis results in decrease in this ratio, which ultimately indicates mitochondrial membrane depolarization [26]. As shown in , the tested peptides produced varying levels of mitochondrial membrane depolarization.…”

Mitochondria Targeted Nanoparticles Potentiate Tumor Chemo‐Phototherapy by Toxic Oxidative Stress Mediated Oxeiptosis

“…However, the reduction of mitochondrial membrane potential makes JC‐10 diffuse out of the mitochondria, change to monomeric form, and make cells fluoresce green. [ 30 ] Compared to the control group, upon the NIR irradiation, the M‐ICG, and M‐ICG‐PTX exhibited decreased red fluorescence and increased green fluorescence (Figure 2b). Since the ratio of red to green fluorescence intensities represents the degree of mitochondria damage, the fluorescence signals of the images were quantified.…”