Nadin Younes1, Bana S Alsahan2, Asmaa J Al-Mesaifri2, Sahar I Da'as3, Gianfranco Pintus4, Amin F Majdalawieh5, Gheyath K Nasrallah1. 1. Biomedical Research Center, QU Health, Qatar University, P.O. Box 2713 Doha, Qatar. 2. Department of Biomedical Science, College of Health Sciences, QU Health, Qatar University, P.O. Box 2713 Doha, Qatar. 3. Department of Human Genetics, Sidra Medicine, P.O. Box 26999, Doha, Qatar. 4. Department of Medical Laboratory Sciences, University of Sharjah, P.O. Box 27272 Sharjah, United Arab Emirates. 5. Department of Biology, Chemistry, and Environmental Sciences, College of Arts and Sciences, American University of Sharjah, P.O. Box 26666 Sharjah, United Arab Emirates.
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.
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.
Authors: Daniel Prantner; Darren J Perkins; Wendy Lai; Mark S Williams; Shruti Sharma; Katherine A Fitzgerald; Stefanie N Vogel Journal: J Biol Chem Date: 2012-10-01 Impact factor: 5.157
Authors: C Adrie; M Bachelet; M Vayssier-Taussat; F Russo-Marie; I Bouchaert; M Adib-Conquy; J M Cavaillon; M R Pinsky; J F Dhainaut; B S Polla Journal: Am J Respir Crit Care Med Date: 2001-08-01 Impact factor: 21.405