从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Summary

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Abstract

功能失调骨骼肌线粒体起到与老化，肥胖和II型糖尿病观察改变的代谢作用。从分离的线粒体制剂线粒体呼吸计测定允许对线粒体功能的药物和能调节代谢蛋白质的作用机制（多个）的评估，以及判定。目前的隔离程序往往需要大量的组织得到必要的透气性测定高品质的线粒体。本文所提出的方法描述了如何高品质纯化线粒体（〜450微克），可以从最小量的小鼠骨骼肌（〜75-100毫克）分离为高通量呼吸测量使用。我们确定了我们的隔离方法产生92.5±2.0％的完整线粒体通过测量柠檬酸合成酶的活性分光光度法。此外，在分离线粒体Western blot分析结果在微弱的表达cytoso的LIC蛋白，GAPDH，和线粒体蛋白质，COXIV的健壮表达。由于没有在分离线粒体的一个突出的GAPDH频带的指示小污染从隔离过程期间非线粒体来源。最重要的是_，O 2的消耗率与微板为基础的技术，并确定用于耦合呼吸计测定显示高度耦合的呼吸控制率（RCR）的测量（RCR;> 6对于所有测定）和功能性线粒体。总之，增加了一个单独的切碎工序和显著减少先前报道的方法的电动机驱动的均化速度已允许的高品质和纯化的线粒体从较少量的小鼠骨骼肌，其导致高度耦合的线粒体，随着高功能呼吸作用的隔离在基于微孔板respirometirc检测。

Introduction

The primary function of mitochondria is to produce ATP from oxidative phosphorylation. However, mitochondria have many other important cellular functions including but not limited to: the production and detoxification of reactive oxygen species, the regulation of cytoplasmic and mitochondrial calcium, organelle trafficking, ionic homeostasis, and involvement in apoptosis^1,2. Therefore, it is not surprising that dysfunctional mitochondria play a role in many disease pathologies, such as aging, neurodegenerative diseases, cardiovascular disease, cancer, obesity, and diabetes^3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies^3-5.

Mitochondrial respiration assays using isolated mitochondria allow for the assessment of electron transport chain and oxidative phosphorylation function, and the determination of mechanism(s) of action of drugs and proteins that modulate metabolism. Mitochondrial isolation procedures exist for multiple tissue and cell types for a variety of species^6,7. However, these procedures often require large quantities of tissue/cells for a high quality mitochondria yield necessary for classic respirometric assays.

Microplate based respirometirc assays allow for high throughput measurements using minimal quantities of isolated mitochondria, often just several µg per well⁸. Therefore, we present a modification of previously published methods⁷ to allow for high quality mitochondria to be isolated from smaller quantities of mouse skeletal muscle for use in microplate based respirometirc assays. In addition, methods are provided to establish the quality of the mitochondrial isolation preparation and the integrity of the mitochondrial membranes. Given that skeletal muscle mitochondria are involved in many pathological conditions, the measurement of O₂ consumption in mechanistically driven studies is becoming more prevalent in biomedical research^9,10.

Protocol

经批准的协议下进行的机构动物护理动物的研究和利用委员会在弗吉尼亚理工学院和州立大学。 1.设置（时间：〜45分钟） 0.25％胰蛋白酶，分离缓冲液为线粒体（IBM）的1和IBM2在37℃水浴中解冻冷冻的商店。 冲洗玻璃器皿和在70％乙醇接着通过高纯度水解剖仪器。 由4部分IBM1稀释1份胰蛋白酶准备从0.25％胰蛋白酶股票0.05％胰蛋白酶溶液。 混合蛋白…

Representative Results

柠檬酸合酶活性充当细胞膜的完整性，因为柠檬酸合酶位于线粒体内膜，并且因此不应该存在于线粒体中具有完整的膜悬浮液的测量。 图1表示非超声处理线粒体样品中的柠檬酸合酶活性带有超声处理相比样品相同的隔离。声波处理线粒体结果在统计学上显著增加柠檬酸合酶活性（P <0.01）。重要的是，以下的隔离线粒体的92.5±2.0％是完整的。 图2描述了</…

Discussion

本文所提出的方法提供了从最小量的小鼠骨骼肌（〜75-100毫克）线粒体分离方法的详细描述。这种隔离方法能够产生高功能，纯粹的线粒体（〜450微克）就证明了_氧气的消耗速率，RCR值，最大的柠檬酸合成酶的活性和免疫蛋白的表达。重要的是，可用于多种respirometirc测定用微孔板基于_O 2的消耗技术，它允许高通量从这个过程中分离线粒体。

骨骼肌线粒体隔离…

Materials

Essentially Fatty	Sigma Aldrich	A6003	N/A
Acid Free- BSA
Tris/HCl	Promega	H5123	N/A
KCL	Sigma Aldrich	P9541	N/A
Tris Base	Promega	H5135	N/A
EDTA	Sigma Aldrich	E6511	N/A
EGTA	Sigma Aldrich	E4378	N/A
Sucrose	Sigma Aldrich	S7903	N/A
D-Mannitol	Sigma Aldrich	63559	N/A
Trypsin-EDTA (0.25%), phenol red	Thermo Scientific	25200-056	N/A
Sodium Chloride White Crystals or Crystalline Powder ≥99.0 %	Fisher Scientific	BP3581	N/A
Sodium dodecyl sulfate	Sigma Aldrich	L3771	N/A
Sodium deoxycholate	Sigma Aldrich	D6750	N/A
Polyoxyethylene (12) nonylphenyl ether, branched	Sigma Aldrich	238651	N/A
Single Edge Razor Blades	Fisher Scientific	12-640	N/A
Falcon- 100 uM Nylon Cell Strainers	Fisher Scientific	352360	N/A
Halt Protease & Phosphatse Inhibitor Cocktail	Thermo Scientific	1861284	N/A
1.5mL microcentrifuge tubes with screw cap	Thermo Scientific	3474	N/A
Zirconium Oxide beads	Fisher Scientific	C9012112	N/A
GAPDH antibody (1D4)	Santa Cruz Biotechnology	sc-59540	N/A
Anti- COXIV antibody	Cell Signaling	4844s	Any mitochondrial inner membrane protein will suffice
Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)	Jackson ImmunoResearh	711-035-152	N/A
Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)	Jackson ImmunoResearh	115-001-003	N/A
Triton-X100	Sigma Aldrich	X100	N/A
Pierce BCA Protein Assay Kit	Thermo Scientific	23225	N/A
Pyruvic Acid, 98%	Sigma Aldrich	107360	Store at 4°C,pH to 7.4 with KOH prior to use in respirometric assay
Succinic Acid	Sigma Aldrich	S9512	Store at room temperature, pH to 7.4 with KOH prior to use in respirometric assay
L(-) Malic Acid, BioXtra, ≥95%	Sigma Aldrich	M6413	Store at room temperature, to 7.4 with KOH prior to use in respirometric assay
L-Glutamic acid	Sigma Aldrich	G1251	Store at room temperature, to 7.4 with KOH prior to use in respirometric assay, to 7.4 with KOH prior to use in respirometric assay
Palmitoyl L-carnitine chloride	Sigma Aldrich	P1645	Store at -20°C
Oligomycin A, ≥ 95% (HPLC)	Sigma Aldrich	75351	Store at -20°C
Carbonyl cyanide 4-(trifluoromethoxy)	Sigma Aldrich	C2920	Store at 2-8°C
phenylhydrazone
≥98% (TLC), powder [FCCP]
Antimycin A from streptomyces sp.	Sigma Aldrich	A8674	Store at -20°C
Adenosine 5′-diphosphate monopotassium salt dehydrate [ADP]	Sigma Aldrich	A5285	Store at -20°C, to 7.4 with KOH prior to use in respirometric assay
Rotenone	Sigma Aldrich	R8875	Store at room temperature