Yüksek Verimli Mikroplate Solunum Ölçümler için Fare İskelet Kası Minimal Miktarları gelen Mitokondri İzolasyonu
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
Fonksiyonel iskelet kası mitokondri yaşlanma, obezite ve Tip II diyabeti olan gözlenen değiştirilmiş metabolizmasında rol oynar. İzole mitokondriyal müstahzarlardan mitokondriyal respirometrik deneyler, ilaç ve metabolizmasını modüle eden proteinlerin hareket mekanizması (ler) in mitokondriyal fonksiyonun değerlendirilmesi, hem de belirlenmesi için izin verir. Güncel izolasyon prosedürleri genellikle Respirometrik deneyleri için gerekli yüksek kaliteli mitokondri verim dokusunun büyük miktarlarda gerektirir. Bu tarifnamede sunulan yöntemler (~ 450 ug), yüksek verimli, solunum ölçümlerde kullanmak için, fare, iskelet kası, minimum miktarlarda (~ 75-100 mg) izole edilebilir ne kadar kaliteli saflaştırılmıştır mitokondri tarif eder. Bizim izolasyon yöntemi spektrofotometrik sitrat sentaz aktivitesini ölçerek 92.5 ± 2.0% bozulmamış mitokondri verimleri belirlenmiştir. Buna ek olarak, izole edilmiş bir mitokondride Western blot analizi, cytoso soluk ekspresyonu ile sonuçlandılic proteini, GAPDH ve mitokondriyal protein, COXIV sağlam ifadesi. İzole mitokondri belirgin bir GAPDH bantın yokluğu, izolasyon prosedürü sırasında olmayan mitokondriyal kaynaklardan çok az kirlenme göstergesidir. En önemlisi, O 2 tüketimi mikro plakalı tabanlı teknoloji ile hızı ve yüksek birleştiğinde gösterir akuple Respirometrik deneyleri için solunum kontrolü oranı (RCR) belirlenmesi ölçümü (RCR, tüm tahliller için> 6) ve fonksiyonel mitokondri. Sonuç olarak, ayrı bir kıyma adım eklenmesi ve önemli ölçüde daha önce bildirilen yöntemin motor tahrikli homojenleştirme hızını azaltarak yüksek fonksiyonlu teneffüs yüksek bağlanmış mitokondri sonuçlanır fare iskelet kası küçük miktarlarda yüksek kalitede ve saflaştırılmış mitokondri izolasyonunu sağladı mikro-bazlı respirometirc deneyleri sırasında.
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
Burada sunulan yöntemler fare iskelet kası minimal miktarlarda (~ 75-100 mg) bir mitokondriyal izolasyon prosedürü ayrıntılı bir açıklamasını sağlar. Bu izolasyon prosedürü O 2 tüketimi oranları, RCR değerleri, maksimal sitrat sentaz aktivitesi ve immunoblotting protein ifadesi ile kanıtlandığı gibi yüksek işleyişi, saf mitokondri (~ 450 mikrogram) elde edebiliyor. Önemli olarak, bu prosedür izole mitokondri, yüksek verimlilik sağlayan mikroplaka göre O2 tüketimi tekno…
Divulgazioni
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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