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.
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。
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 apoptosis1,2. 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 diabetes3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies3-5.
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 species6,7. 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 research9,10.
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…
The authors have nothing to disclose.
The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.
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 |