높은 처리량 마이크로 호흡 측정을위한 마우스 골격근의 최소 수량에서 미토콘드리아의 분리
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
역기능 골격근의 미토콘드리아 노화, 비만과 형 당뇨병으로 관찰 변경된 대사의 역할을한다. 절연 미토콘드리아 제제로부터 미토콘드리아 호흡계 분석법은 약물 대사를 조절하는 단백질의 작용기구 (S)의 미토콘드리아 기능의 평가뿐만 아니라, 판정이 가능. 현재 분리 절차는 종종 호흡계 분석에 필요한 고품질 미토콘드리아를 수득 조직 대량을 요구한다. 본원에 제시된 방법 (~ 450 μg의)는 높은 처리량 호흡 측정에 사용하기 위해 마우스 골격근의 최소량 (~ 75-100 mg)을 분리 할 수있는 방법을 고품질로 정제 미토콘드리아 설명한다. 우리는 우리의 분리 방법은 분광 시트르산 합성 효소 활성을 측정하여 92.5 ± 2.0 %의 미토콘드리아 손상을 산출 것으로 판정. 또한, 고립 된 미토콘드리아의 웨스턴 블롯 분석은 cytoso의 희미한 식의 결과LIC 단백질, GAPDH 및 미토콘드리아 단백질, COXIV의 강력한 표현. 단리 된 미토콘드리아에 띄는 GAPDH 밴드의 부재는 분리 과정 동안 비 미토콘드리아 소스로부터 조금 오염을 나타낸다. 가장 중요한 것은, O (2) 소비 마이크로 플레이트 기반 기술과 속도와 고도의 결합을 보여줍니다 결합 호흡계 분석을위한 호흡 조절 비율 (RCR) 결정의 측정 (RCR을, 모든 분석을위한> 6) 및 기능 미토콘드리아. 결론적으로, 별도의 닦지 단계 첨가 및 상당히 이전에보고 된 방법의 모터 구동 균질화 속도를 감소시키는 것은 높은 기능 호흡 고도로 결합 미토콘드리아 결과 마우스 골격근의 소량으로부터 고품질 정제 미토콘드리아의 분리를 허용했다 마이크로 기반 respirometirc 분석 중.
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
본원에 제시된 방법은 마우스 골격근의 최소량 (~ 75-100 mg)을 미토콘드리아로부터 분리 절차의 상세한 설명을 제공한다. 이 분리 절차는 O 2 소비율, RCR 값, 최대 시트 레이트 신타 제 활성 및 면역에서 단백질 발현에 의해 입증되는 바와 같이 높은 작동, 순수한 미토콘드리아 (~ 450 μg의)을 수득 할 수있다. 중요한 것은,이 과정에서 분리 된 미토콘드리아는 높은 처리량을 가능 마이크로 기반…
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 |
Riferimenti
- Brand, M., Nicholls, D. Assessing mitochondrial dysfunction in cells. Biochem. J. 435, 297-312 (2011).
- Nicholls, D. G., Ferguson, S. . Bioenergetics. , (2013).
- Nunnari, J., Suomalainen, A. Mitochondria: in sickness and in health. Cell. 148, 1145-1159 (2012).
- Lauri, A., Pompilio, G., Capogrossi, M. C. The mitochondrial genome in aging and senescence. Ageing Res. Rev. 18, 1-15 (2014).
- Dube, J. J., et al. Effects of acute lipid overload on skeletal muscle insulin resistance, metabolic flexibility, and mitochondrial performance. Am. J. Physiol. Endocrinol. Metab. 12, 1117-1124 (2014).
- Fernandez-Vizarra, E., Lopez-Perez, M. J., Enriquez, J. A. Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells. Methods (San Diego, Calif). 26, 292-297 (2002).
- Frezza, C., Cipolat, S., Scorrano, L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat. Protoc. 2, 287-295 (2007).
- Rogers, G. W., et al. High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PloS one. 6, e21746 (2011).
- Guarino, R. D., et al. Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration. Biotechnol. and Bioeng. 86, 775-787 (2004).
- Will, Y., Hynes, J., Ogurtsov, V. I., Papkovsky, D. B. Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes. Nat. Protoc. 1, 2563-2572 (2007).
- Hulver, M. W., et al. Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans. Cell Metab. 2, 251-261 (2005).
- Frisard, M. I., et al. Toll-like receptor 4 modulates skeletal muscle substrate metabolism. Am. J. Physiol. Endocrinol. Metab. 298, e988-e998 (2010).
- Chance, B., Williams, G. R. The respiratory chain and oxidative phosphorylation. Adv. Enzymol. Relat. Subjects of Biochem. 17, 65-134 (1956).
- Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. Mitochondrial isolation from skeletal muscle. JoVE. , (2011).
- Gross, V. S., et al. Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization. Anal. Biochem. 418, 213-223 (2011).
- Krieger, D. A., Tate, C. A., McMillin-Wood, J., Booth, F. W. Populations of rat skeletal muscle mitochondria after exercise and immobilization. J. Appl. Physiol.: Respir., Envir. and Ex. Physiol. 48, 23-28 (1980).
- Asmann, Y. W., et al. Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia. Diabetes. 55, 3309-3319 (2006).
- Lanza, I. R., Nair, K. S. Functional assessment of isolated mitochondria in vitro. Methods Enzymol. 457, 349-372 (2009).
