उच्च Throughput Microplate श्वसन माप के लिए माउस कंकाल की मांसपेशी की कम मात्रा से माइटोकॉन्ड्रिया के अलगाव
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
बेकार कंकाल की मांसपेशी माइटोकॉन्ड्रिया उम्र बढ़ने, मोटापा और टाइप टू मधुमेह के साथ मनाया बदल चयापचय में एक भूमिका निभाते हैं। पृथक माइटोकॉन्ड्रियल तैयारी से माइटोकॉन्ड्रियल respirometric assays दवाओं और चयापचय को मिलाना है कि प्रोटीन की कार्रवाई की व्यवस्था (एस) के माइटोकॉन्ड्रियल समारोह का आकलन, साथ ही दृढ़ संकल्प के लिए अनुमति देते हैं। वर्तमान अलगाव प्रक्रियाओं अक्सर respirometric assays के लिए आवश्यक उच्च गुणवत्ता माइटोकॉन्ड्रिया उपज के लिए ऊतक की एक बड़ी मात्रा की आवश्यकता होती है। इस के साथ साथ प्रस्तुत तरीकों (~ 450 माइक्रोग्राम) उच्च throughput सांस की माप में उपयोग के लिए माउस कंकाल की मांसपेशी की न्यूनतम मात्रा (~ 75-100 मिलीग्राम) से अलग किया जा सकता है कि कैसे उच्च गुणवत्ता शुद्ध माइटोकॉन्ड्रिया का वर्णन है। हम अपने अलगाव विधि spectrophotometrically साइट्रेट synthase गतिविधि को मापने के द्वारा 92.5 ± 2.0% बरकरार माइटोकॉन्ड्रिया कि पैदावार निर्धारित की। इसके अलावा, अलग-थलग माइटोकॉन्ड्रिया में पश्चिमी धब्बा विश्लेषण cytoso के बेहोश अभिव्यक्ति में हुईएलआईसी प्रोटीन, GAPDH, और mitochondrial प्रोटीन, COXIV के मजबूत अभिव्यक्ति। पृथक माइटोकॉन्ड्रिया में एक प्रमुख GAPDH बैंड के अभाव अलगाव की प्रक्रिया के दौरान गैर-माइटोकॉन्ड्रियल स्रोतों से थोड़ा संदूषण का संकेत है। सबसे महत्वपूर्ण बात, हे 2 खपत सूक्ष्म प्लेट आधारित तकनीक के साथ दर और अत्यधिक युग्मित दिखाता युग्मित respirometric assays के लिए श्वसन नियंत्रण अनुपात (रेसकोर्स रोड) का निर्धारण करने का माप (रेसकोर्स रोड, सब assays के लिए> 6) और कार्यात्मक माइटोकॉन्ड्रिया। अंत में, एक अलग नख़रेबाज़ कदम के अलावा और काफी पहले से सूचना दी विधि की मोटर चालित homogenization गति को कम करने के लिए उच्च समारोह के साथ साँस लेते हैं कि अत्यधिक युग्मित माइटोकॉन्ड्रिया में यह परिणाम है कि माउस कंकाल की मांसपेशी की छोटी मात्रा से उच्च गुणवत्ता और शुद्ध माइटोकॉन्ड्रिया के अलगाव की अनुमति दी है microplate आधारित respirometirc assays के दौरान।
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 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.
Protocol
Representative Results
Discussion
इस के साथ साथ प्रस्तुत तरीकों माउस कंकाल की मांसपेशी की न्यूनतम मात्रा (~ 75-100 मिलीग्राम) से एक माइटोकॉन्ड्रियल अलगाव की प्रक्रिया का विस्तृत विवरण प्रदान करते हैं। इस अलगाव की प्रक्रिया के ओ 2 खपत की…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.
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 |
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