title: Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement.
abstract: The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key toin vitrostudies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renaultet al., 2013). In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is "Liver mitochondria isolation" in which the liver is dissected from mice to obtain mitochondria. "Mitochondria labeling with JC-1 and size fractionation" is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the "Real-time MOMP measurements" protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determinein vitrothe role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renaultet al., 2015).
authors.affiliation: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; The Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; Department of Dermatology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; Helmholtz Centre for Infection Research, Braunschweig, Germany; Max Planck Institute for Infection Biology, Charitéplatz 1, Campus Charité Mitte, Berlin, Germany.
authors.forename: Thibaud T
authors.initials: TT
authors.lastname: Renault
authors.affiliation: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA.
authors.forename: Mark P A
authors.initials: MP
authors.lastname: Luna-Vargas
authors.affiliation: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; The Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; Department of Dermatology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA.
authors.forename: Jerry E
authors.initials: JE
authors.lastname: Chipuk