小鼠肝脏线粒体分离、大小分级和实时外膜通透性（MOMP）测量

Thibaud T. Renault; Mark P.A Luna-Vargas; Jerry E. Chipuk

doi:10.21769/BioProtoc.1892

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Peer-reviewed

Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement

小鼠肝脏线粒体分离、大小分级和实时外膜通透性（MOMP）测量

TR Thibaud T. Renault email

Mark P.A Luna-Vargas

Jerry E. Chipuk email

发布: 2016年08月05日第6卷第15期 DOI: 10.21769/BioProtoc.1892 浏览次数: 12340

评审: Pia GiovannelliAnonymous reviewer(s)

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Cover of Molecular Cell, featuring study using the protocol.

Jan 2015

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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 to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et 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 determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al., 2015).

Keywords: Apoptosis (细胞凋亡)

Mitochondria (线粒体)

BCL-2 Family (Bcl-2家族)

MOMP (MOMP)

Mitochondrial Fractionation (线粒体的分离)

Materials and Reagents

50 ml conical centrifuge tube (Santa Cruz Biotechnology, catalog number: sc-200251 )
Petri dish, 100 x 15 mm (Fisher Scientific, catalog number: FB0875712 )
15 ml conical centrifuge tube (Santa Cruz Biotechnology, catalog number: sc-200250 )
1.5 ml Micro centrifuge tube (USA Scientific, catalog number: 1615-5510 )
Gravity chromatography column (Thermo Fisher Scientific, Pierce^TM, catalog number: 29920 )
Pasteur pipet (Santa Cruz Biotechnology, catalog number: sc-204537 )
96-well plate, flat bottom, black polystyrene (Corning, Costar^TM, catalog number: 3915 )
C57BL/6 mice (Charles River Laboratories, catalog number: 027 )
1x phosphate buffered saline (PBS), pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄) (Fisher Scientific, catalog number: BP24384 )
Trehalose (Sigma-Aldrich, catalog number: 1673715 )
Sucrose (Sigma-Aldrich, catalog number: S0389 )
[4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid]-KOH (HEPES) (Sigma-Aldrich, catalog number: H0527 )
KCl (Sigma-Aldrich, catalog number: P9541 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
Ethyleneglycoltetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
Bovine serum albumin-fraction V (Sigma-Aldrich, catalog number: A9418 )
Protease inhibitor cocktail (Thermo Fisher Scientific, HALT^TM, catalog number: 78430 )
Sepharose CL-2B resin (Sigma-Aldrich, catalog number: CL2B300 )
5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Thermo Fisher Scientific, catalog number: T3168 )
Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich, catalog number: C2920 )
Recombinant BAX [purified using the IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system (New England Biolabs, catalog number: E6901S )]
β-octylglucoside (OG) (Sigma-Aldrich, catalog number: O8001 )
Dimethylsulfoxide (DMSO) (Fisher Scientific, catalog number: BP231-100 )
Magnesium Chloride Hexahydrate (Fisher Scientific, catalog number: M33-500 )
Trehalose isolation buffer (TIB) (see Recipes)
JC-1 loading buffer (see Recipes)
Liposome buffer (see Recipes)

Equipment

Dissection tools (scissors, scalpel, forceps)
Razor blade (Daigger Scientific, catalog number: EF7281A )
15 ml Potter-Elvehjem dounce homogenizer (Omni International, catalog number: 07-358044 )
Swing bucket centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM Sorvall^TM, model: Legend XTR )
Spectrophotometer (Fisher Scientific, GE Healthcare Ultrospec^TM, model: Ultrospec 7000 )
Water bath (Fisher Scientific, IsoTemp^TM, model: 215 )
Tabletop centrifuge (Thermo Fisher Scientific, Sorvall^TM Legend^TM, model: Micro 21 )
Fluorescence plate reader (Biotek, model: Synergy H1 )

Procedure

English

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如何引用

Renault, T. T., Luna-Vargas, M. P. and Chipuk, J. E. (2016). Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement. Bio-protocol 6(15): e1892. DOI: 10.21769/BioProtoc.1892.