Выделение митохондрий из минимальных количествах мыши скелетных мышц для измерения Высокая пропускная микропланшетов дыхания
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
Неблагополучные скелетные мышечные митохондрии играют важную роль в обмене веществ наблюдается измененной со старением, ожирения и сахарного диабета II типа. Митохондриальные respirometric анализы из изолированных препаратах митохондрий позволяют для оценки функции митохондрий, а также определения механизма (ов) действия лекарственных средств и белков, которые модулируют метаболизм. Текущие процедуры изоляции часто требуют большого количества ткани, чтобы получить высокие митохондрии качества, необходимые для respirometric анализов. Методы, представленные здесь, описывают, как высокое качество очищают митохондрии (~ 450 мкг) может быть выделен из минимальных количествах (~ 75-100 мг) мыши скелетных мышц для применения в дыхательных измерений с высокой пропускной способностью. Мы решили, что наш метод изоляции дает 92,5 ± 2,0% нетронутыми митохондрии путем измерения активности цитрат-синтазы спектрофотометрически. Кроме того, Вестерн-блот анализ в изолированной митохондрии в результате слабый экспрессии cytosoLIC белок, GAPDH и надежный выражение белка митохондрий, COXIV. Отсутствие видного GAPDH полосы в изолированных митохондриях свидетельствует о небольшом загрязнения от не-митохондриальных источников во время процедуры изоляции. Самое главное, что измерение O 2 скорости потребления с технологией, основанной микро-пластины и определения дыхательный коэффициент (РКП) для связанных respirometric анализов показывает весьма сочетании (RCR;> 6 для всех анализов) и функциональные митохондрии. В заключение, добавление отдельной стадии мясорубки и значительно снижая приводом двигателя скорость гомогенизации сообщалось ранее метода позволило изоляции высокого качества и очищенной митохондрий от небольших количеств мыши скелетных мышц, что приводит к высокой связанных митохондрий, что дышать с высокой функции во микропланшета на основе respirometirc анализов.
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
Методы, представленные здесь, обеспечивают подробное описание митохондриальной процедуры выделения из минимальных количествах (~ 75-100 мг) мыши скелетных мышц. Эта процедура изоляции способен дать высокую функционирование митохондрий, чистый (~ 450 мкг), о чем свидетельствует вывода 2</s…
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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