Name: isolation of mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements
Uploaded: 2015-11-13T14:00:00.000+00:00
Duration: 10 min 12 s
Description: Watch this Scientific Journal Video about Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements at JoVE.com

The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.

동물 실험 기관 동물 관리에 의해 승인 된 프로토콜에 따라 수행 버지니아 폴리 테크닉 연구소 및 주립 대학에서위원회를 사용했다. 1. 설정 (시간 : ~ 45 분) <ol><li> 37 ° C의 물을 욕조에 미토콘드리아 (IBM) 1 IBM2 0.25 % 트립신, 절연 버퍼의 해동 냉동 저장합니다. </li><li> 유리 및 고순도 물 다음 70 % 에탄올에 해부 악기를 씻어. </li><li> 4 부 IBM1 1 부 트립신을 희석하여 0.25 % 트립신 재고에서 0.05 % 트립신 솔루션을 준비합니다. </li><li> 스크류 캡과 1.5 ml의 microcentrifuge 관에 : (100 비율 1) 세포 용해 버퍼와 단백질 분해 효소와 포스 파타 아제 억제제 칵테일을 섞는다. </li><li> 분취 량 5 ml를 15 ml의 플라스틱 튜브에 0.05 % 트립신 (5 ㎖ / 마우스). </li><li> 설정된 모터 80 RPM으로 조직 균질기를 구동. </li></ol> 골격근 미토콘드리아 2. 분리 (시간 : ~ 90 분) <ol><li> CO 2로 마우스를 안락사 </su자궁 경부 전위 다음에 B&gt; 흡입,. </li><li> 다음 단계에 설명 된대로 가자미근 (~ 75-100 mg의 총)을 포함하는 대퇴사 두근과 비복근 근육, 붉은 근육을 제거합니다 : <ol><li> 마우스쪽으로 피부 껍질. </li><li> 대퇴사 두근 원점을 통해 지방 패드를 제거합니다. 뾰족한 가위로 슬개골에 부착 된 대퇴사 두근 건을 잘라. 참고 : 대퇴사 두근은 근위 대퇴골의 앞쪽 부분에 자사의 해부학 적 위치로 식별됩니다. </li><li> 뼈에서 대퇴사 두근을 해방, 대퇴 동맥을 피하면서 천천히, 뼈와 대퇴사 두근의 건막을 싹둑. 대퇴사 두근 근육을 해방하고 냉장 PBS의 대퇴사 두근 근육을 배치 대퇴골에 원점에서 뾰족한 가위로 힘줄을 잘라. </li><li> 가위로 대퇴사 두근을 통해 눈에 보이는 지방 조직을 제거합니다. 그래서 대퇴골 위에 놓인 된 근육의 부분이 U를 향하도록 통해 대퇴사 두근 플립피. 패닝의 움직임에 집게로 대퇴사 두근 근육을 엽니 다. 냉각 IBM1 5 mL를 넣은 비커에 뾰족한 가위와 장소 각 로브에서 두 개의 눈에 보이는 붉은 근육 부분을 제거합니다. 참고 : 대퇴사 근육 각 엽의 측면 가장자리 근처에있는 붉은 근육의 스트립 두 엽으로 나타납니다. </li><li> 뾰족한 가위로 아킬레스 건을 덮는 피부를 잘라 마우스 향해 피부를 벗겨. 노출 아킬레스 건을 잘라 마우스의 몸으로 근육 껍질. </li><li> 비복근을 해방하고 냉장 PBS에 그 힘줄에 부착 된 비복근을 배치 할 뾰족한 가위로 대퇴골의 외측과 내측 과두의 힘줄을 잘라. </li><li> 이상 비복근 플립과 가자미 근육을 벗겨 차게 IBM1 5 mL를 포함하는 비커에 넣습니다. 팬 비복근을 엽니 다. 뾰족한 가위로 3 종류의 시각적 붉은 근육 부분 (두 측면과 하나의 내측 표면 빨간색 스트립)를 제거S 및 냉장 IBM1 5 ㎖을 함유하는 비이커에 옮긴다. </li></ol></li><li> (단계 2.2에 기재된 바와 같이) 마우스의 다른 레그에서 적색 근육의 또 다른 ~ 75 내지 100 mg의 분리하고 프로테아제 및 포스파타제 억제제 칵테일 및 세포 용해 완충액 (50 mM Tris-HCl, 1 1.5 ㎖의 마이크로 원심 튜브에 배치 mM의 EDTA, 150 mM의 염화나트륨, 1 % 소듐 도데 실 설페이트, 0.5 % 나트륨 데 옥시 콜레이트, 1 % 폴리 옥시 에틸렌 (9) 노닐 페닐이. 즉시 블 롯팅 웨스턴에서 나중에 사용하기 위해 액체 질소에서이 제 2 샘플을 플래시 동결 분지 (단계 6.1 참조). </li><li> 큰 양동이에 얼음에 미리 냉장, 평면 플라스틱 표면을 놓습니다. 플라스틱 표면에 IBM1 한 방울을 붓고 IBM1 방울의 단면 붉은 근육 (단계 2.2.4과 2.2.7)을 모두 배치 핀셋을 사용합니다. 변경 면도기 3 단일 에지 면도날마다 40 초를 사용하여 2 분 동안 근육 조직을 말하다. </li><li> 제 위에 플라스틱 표면을 유지함으로써 새로운 IBM1 5 ㎖와 새로운 비이커 다진 조직을 이동시켜전자 비커와 면도날로 비커에 근육을 긁어. 이 솔루션을 가지고 50 ML 원뿔 튜브에 배치 된 100 μm의 셀 스트레이너를 통해 배출. </li><li> 섬세한 작업 와이퍼와 조직을 가릴 후 0.05 % 트립신 용액 5 ㎖로 옮긴다. 작업 와이퍼에서 조직을 제거하는 핀셋을 사용합니다. </li><li> 0.05 % 트립신 용액에서 30 분 동안 얼음에 근육 조직을 배양한다. </li><li> 4 ℃에서 3 분 동안 200 XG에 15 ML 원뿔 튜브 포함 트립신 / 근육 혼합물을 스핀. </li><li> 폐기물 용기에 트립신 뜨는을 붓고 얼음 3 ㎖ 차가운 IBM1와 펠렛을 재현 탁. 45 ㎖의 유리 균질 튜브에 조직을 전송합니다. 나머지 샘플을 수집하고 유리 균질 관이를 추가하는 또 다른 1.5 ml의 IBM1에 15 ml의에게 원뿔 튜브를 씻어. </li><li> 유리 균질이 비커 내에서 최소한의 이동 때문에 얼음 비커 또는 플라스틱 용기 충전 중간에 유리 균질 튜브를 놓습니다. </li><li&gt; 80 rpm으로 10 패스와 모터 구동 조직 균질에 부착 된 PTFE와 유 봉, 조직 / IMB1 혼합물을 균질화. ~ 2 초 동안 각 패스의 바닥을 잡으십시오. </li><li> 새로운 15ml의 원뿔형 튜브에 조직 파쇄 옮기고 IBM1 6.5 ml의 균질화 유리 튜브를 헹군다. 조직 균질 물을 포함하는 15ml의 원뿔형 튜브에 IBM1 붓는다. </li><li> 4 ° C에서 10 분 동안 700g로 15 ml의 원추형 튜브 스핀. 부드럽게 유리 고강도 원심 분리기 튜브에 뜨는을 부어 펠렛을 폐기합니다. </li><li> 4 ℃에서 10 분 동안 8,000 XG에서 위의 단계에서 뜨는 스핀. </li><li> IBM1 상층 액을 제거하고 천천히 IBM2 500 μl를 추가하여 펠렛을 다시 일시 중지합니다. 피펫 팁의 끝을 잘라 부드럽게 혼합 운동을 교반 IBM2에 펠릿을 균질화. 펠렛가 완전히 정지 한 후 혼합물에 IBM2의 또 다른 4.5 ML을 추가합니다. T와 같은 더 큰 펠릿 조각을 파괴하면서 과도한 분쇄를 피하십시오 : 주자신은 미토콘드리아의 세포막에 손상을 줄 수 있습니다. </li><li> 4 ° C에서 10 분 동안 8,000 XG에 IBM2 / 조직 파쇄 스핀. 주의 :이 단계는 총 단백질 함량에 기여할 수있다 잔류 BSA 4 단계 이후에 격리 미토콘드리아 단백질보다 정확한 정량 깨끗한 고강도 원심 분리 튜브에서 반복 될 수있다. </li><li> 부드럽게 뜨는 및 제거,하지만 완전히 차단의 포인트 피펫 팁과 IBM2의 두 25 μL 단위를 추가하여 펠렛을 다시 일시 중지합니다. 부드럽게 저어 IBM2 25 μL를 각각 첨가 한 후 펠렛을 섞는다. 얼음이 미토콘드리아 주식을 놓습니다. </li></ol> 전체 조직 해물 3. 균질화 (시간 : ~ 45 분; 단계에서 수행 할 수 있습니다 7) <ol><li> 즉, 단계 230에서 액체 질소에 넣고에서 근육을 제거하고 완전 해동 될 때까지 실온에서 배양한다. </li><li> 뾰족한 가위로 얼음 ~ 30 초 동안 조직을 말하다. </li><li> Zirco의 두 100 μL 개만 추가다진 조직을 포함 단계 3.2에서 스크류 캡으로 1.5 ml의 microcentrifuge 관에 천년 산화물 비즈. 4-6 (매체 설정)의 속도 설정에서 3 개의 5 분주기를 이용하여 비드 밀 조직 균질 기에서 균질화 조직. </li><li> 4 ℃에서 10 분 동안 12,000 XG에 단계 3.3에서 혼합물을 스핀. 1.5 ml의 microcentrifuge 관에 스핀 및 전송 후 1,000 μL 피펫 팁을 사용하여 상층 액을 제거합니다. 펠렛을 취소하고 얼음에 뜨는을 배치합니다. </li></ol> 4. 단백질 결정 (시간 : ~ 30 분) <ol><li> 제조업체의 사양에 따라 BCA 단백질 분석 키트를 사용하여 전체 조직 용 해물 (단계 3.4) 및 미토콘드리아 스톡 (단계 2.17)의 단백질 농도를 결정한다. </li></ol> 5. 미토콘드리아 막 무결성; 구연산 합성 효소 (CS) 활동 (시간 : ~ 15 분) <ol><li> 고순도 물에 미토콘드리아 주식의 20 희석 : 두 개의 1을 확인합니다. 더하다하나의 샘플에 트리톤 100-X의 0.1 % 및 낮은 설정 (765 와트, 2의 진폭)에 그것을 초음파 처리. 얼음에 다른 희석 둡니다. 초음파 후 얼음에 샘플을 돌려줍니다. </li><li> 시트 레이트 신타 제와 분석을 수행 모두 비 초음파 처리하고, 이전에 기술 된 바와 같이 (11, 12)을 분광 분석을 이용하여 미토콘드리아 희석액 초음파. </li><li> 다음 식을 사용하여 그대로 미토콘드리아 막의 비율을 결정 : (비 초음파 CS 활성 ÷ 초음파 CS 활성) * 100 </li></ol> N은 100- (퍼센트 위에서 달성) 6. 미토콘드리아 격리 품질; 웨스턴 블로 팅 (시간 : 실험실 프로토콜에 따라) <ol><li> 이전 12 설명 된대로 전체 조직 해물과 격리 된 미토콘드리아에 웨스턴 블로 팅을 수행합니다. 참고 : GAPDH에 대한 사용 차 항체 (1 : 1000 희석 5 %의 BSA / TBS-T) 및 COXIV (1 : 1,000 희석 5 % 무 지방우유 / TBS-T 각각 반 염소, 토끼 이차 항체 (1 다음에) : 10,000). </li></ol> 7. 호흡계 분석 실행 (시간 : ~ 90 분) <ol><li> 전술 한 바와 같이 마이크로 기반 O 2 소비량 측정 기술을 이용하여 원하는 분석을 수행 호흡계 (컴패니언 문서 및 로저스 외 8 참조). </li><li> 주 4O (RCR)에 의해 상태 3 분할 또는 주 4O 의해 주 3U 나누어 호흡 조절 비율 (RCR)를 결정한다. RCR을 결정할 때 포인트 - 투 - 포인트 흔적에서 중간 (평균) 점, 또는 가장 높은 (주 3), 저렴한 가격 (주 섭씨 40도)를 사용합니다. </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> JoVE. , (2011).</li><li>Gross, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+functional+mitochondria+from+rat+kidney+and+skeletal+muscle+without+manual+homogenization.">Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization.</a> Anal. Biochem. 418, 213-223 (2011).</li><li>Krieger, D. A., Tate, C. A., McMillin-Wood, J., Booth, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Populations+of+rat+skeletal+muscle+mitochondria+after+exercise+and+immobilization.">Populations of rat skeletal muscle mitochondria after exercise and immobilization.</a> J. Appl. Physiol.: Respir., Envir. and Ex. Physiol. 48, 23-28 (1980).</li><li>Asmann, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Diabetes. 55, 3309-3319 (2006).</li><li>Lanza, I. R., Nair, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+isolated+mitochondria+in+vitro.">Functional assessment of isolated mitochondria in vitro.</a> Methods Enzymol. 457, 349-372 (2009).</li></ol>

George Rogers is an employee of Seahorse Bioscience that produces the instrument for which this protocol was modified.

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...

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 &#181;g 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.

<table><tbody><tr><td>Essentially Fatty</td><td>Sigma Aldrich</td><td>A6003</td><td>N/A</td></tr><tr><td>Acid Free- BSA</td><td></td><td></td><td></td></tr><tr><td>Tris/HCl</td><td>Promega</td><td>H5123</td><td>N/A</td></tr><tr><td>KCL</td><td>Sigma Aldrich</td><td>P9541</td><td>N/A</td></tr><tr><td>Tris Base</td><td>Promega</td><td>H5135</td><td>N/A</td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E6511</td><td>N/A</td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td>N/A</td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S7903</td><td>N/A</td></tr><tr><td>D-Mannitol</td><td>Sigma Aldrich</td><td>63559</td><td>N/A</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Thermo Scientific</td><td>25200-056</td><td>N/A</td></tr><tr><td>Sodium Chloride&lt;br/&gt; White Crystals or Crystalline Powder&lt;br/&gt; &amp;ge;99.0 %</td><td>Fisher Scientific</td><td>BP3581</td><td>N/A</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma Aldrich</td><td>L3771&amp;nbsp;</td><td>N/A</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma Aldrich</td><td>D6750&amp;nbsp;</td><td>N/A</td></tr><tr><td>Polyoxyethylene (12) nonylphenyl ether, branched</td><td>Sigma Aldrich</td><td>238651</td><td>N/A</td></tr><tr><td>Single Edge Razor Blades</td><td>Fisher Scientific</td><td>12-640</td><td>N/A</td></tr><tr><td>Falcon- 100 uM Nylon Cell Strainers</td><td>Fisher Scientific</td><td>352360</td><td>N/A</td></tr><tr><td>Halt Protease &amp;amp; Phosphatse Inhibitor Cocktail</td><td>Thermo Scientific</td><td>1861284</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes with screw cap</td><td>Thermo Scientific</td><td>3474</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Zirconium Oxide beads</td><td>Fisher Scientific</td><td>C9012112</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>GAPDH antibody (1D4)</td><td>Santa Cruz Biotechnology</td><td>sc-59540</td><td>N/A</td></tr><tr><td>Anti- COXIV antibody</td><td>Cell Signaling</td><td>4844s</td><td>Any mitochondrial inner membrane protein will suffice</td></tr><tr><td>Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>711-035-152</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>115-001-003</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Triton-X100</td><td>Sigma Aldrich</td><td>X100</td><td>N/A</td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Scientific</td><td>23225</td><td>N/A</td></tr><tr><td>Pyruvic Acid, 98%</td><td>Sigma Aldrich</td><td>107360</td><td>Store at 4 &amp;deg;C,pH to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>Succinic Acid</td><td>Sigma Aldrich</td><td>S9512</td><td>Store at room temperature, pH to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>L(-) Malic Acid, BioXtra, &amp;ge;95%</td><td>Sigma Aldrich</td><td>M6413</td><td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>L-Glutamic acid</td><td>Sigma Aldrich</td><td>G1251</td><td>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</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Palmitoyl L-carnitine chloride</td><td>Sigma Aldrich</td><td>P1645</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Oligomycin A, &amp;ge; 95% (HPLC)</td><td>Sigma Aldrich</td><td>75351</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)</td><td>Sigma Aldrich</td><td>C2920</td><td>Store at 2-8 &amp;deg;C</td></tr><tr><td>phenylhydrazone</td><td></td><td></td><td></td></tr><tr><td>&amp;ge;98% (TLC), powder [FCCP]</td><td></td><td></td><td></td></tr><tr><td>Antimycin A from streptomyces sp.</td><td>Sigma Aldrich</td><td>A8674</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate monopotassium salt dehydrate [ADP]</td><td>Sigma Aldrich</td><td>A5285</td><td>Store at -20 &amp;deg;C, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Rotenone</td><td>Sigma Aldrich</td><td>R8875</td><td>Store at room temperature</td></tr></tbody></table>

isolation of mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements

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 수​​있는 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&gt; 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.

시트 레이트 신타 제 활성은 시트 레이트 신타이 양막 미토콘드리아 현탁액으로 존재해서는 안 따라서 미토콘드리아 내막에 위치되고, 이후 막 무결성을위한 수단으로서 기능한다. (1)이 초음파에 비해 비 초음파 미토콘드리아 시료에 시트 레이트 신타 제 활성을 나타내는 도표 같은 격리에서 샘플. 시트 레이트 신타 제 활성의 유의 한 증가 미토콘드리아 결과 (P &lt;0.01)?...

Watch this Scientific Journal Video about Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements at JoVE.com

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

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.

높은 처리량 마이크로 호흡 측정을위한 마우스 골격근의 최소 수량에서 미토콘드리아의 분리

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

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Biology

12.7K 조회수.  Virginia Tech. 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.

비디오: Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

높은 처리량 마이크로 호흡 측정을위한 마우스 골격근의 최소 수량에서 미토콘드리아 격리 사용

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

연구

발생학

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

골격 근육 미토콘드리아의 단백질 수입 능력 측정

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

생화학

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

경피적 바늘 생검에 의해 획득 골격근 조직에서 고립 된 미토콘드리아의 준비 및 호흡계 평가

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

의학

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

생물학

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

골격근에서 Mitochondrial 절연

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

골격근에서 Progenitors의 정화

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

<meta charset="utf8"></head><body>고품질 생체 에너지 연구를 위한 설치류의 미토콘드리아 분리

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

<meta charset="utf8"></head><body>저자 스포트라이트: 심부전 및 생체 에너지학에서 미토콘드리아 인산칼슘의 역할 밝혀내기

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

설치류 심장 조직으로부터 강력한 미토콘드리아 분리 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

미토콘드리아 절연 마우스 중심부에 투과율 전환 기공 영업의 다중 매개 변수 측정

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

높은 처리량 마이크로 호흡 측정을위한 마우스 골격근의 최소 수량에서 미토콘드리아의 분리

요약

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이 비디오의 챕터

높은 처리량 마이크로 호흡 측정을위한 마우스 골격근의 최소 수량에서 미토콘드리아 격리 사용

골격 근육 미토콘드리아의 단백질 수입 능력 측정

경피적 바늘 생검에 의해 획득 골격근 조직에서 고립 된 미토콘드리아의 준비 및 호흡계 평가

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

골격근에서 Mitochondrial 절연

골격근에서 Progenitors의 정화

설치류 심장 조직으로부터 강력한 미토콘드리아 분리

설치류 심장 조직으로부터 강력한 미토콘드리아 분리

설치류 심장 조직으로부터 강력한 미토콘드리아 분리

미토콘드리아 절연 마우스 중심부에 투과율 전환 기공 영업의 다중 매개 변수 측정