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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Essentially Fatty</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A6003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
			<td>Acid Free- BSA</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 
			White Crystals or Crystalline Powder 
			&#8805;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&#160;</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma Aldrich</td>
			<td>D6750&#160;</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 rowspan="2">Halt Protease &#38; Phosphatse Inhibitor Cocktail</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">1861284</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">1.5mL microcentrifuge tubes with screw cap</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">3474</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Zirconium Oxide beads</td>
			<td rowspan="2">Fisher Scientific</td>
			<td rowspan="2">C9012112</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</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 rowspan="2">Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">711-035-152</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">115-001-003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</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&#160;</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&#176;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, &#8805;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 rowspan="2">L-Glutamic acid</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">G1251</td>
			<td rowspan="2">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>
		</tr>
		<tr>
			<td>Palmitoyl L-carnitine chloride</td>
			<td>Sigma Aldrich</td>
			<td>P1645</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Oligomycin A, &#8805; 95% (HPLC)</td>
			<td>Sigma Aldrich</td>
			<td>75351</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)</td>
			<td rowspan="3">Sigma Aldrich</td>
			<td rowspan="3">C2920</td>
			<td rowspan="3">Store at 2-8&#176;C</td>
		</tr>
		<tr>
			<td>phenylhydrazone</td>
		</tr>
		<tr>
			<td>&#8805;98% (TLC), powder [FCCP]</td>
		</tr>
		<tr>
			<td>Antimycin A from streptomyces sp.</td>
			<td>Sigma Aldrich</td>
			<td>A8674</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td rowspan="2">Adenosine 5&#8242;-diphosphate monopotassium salt dehydrate [ADP]</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A5285</td>
			<td rowspan="2">Store at -20&#176;C, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
		</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 ...

isolation-mitochondria-from-minimal-quantities-mouse-skeletal-muscle

Research

JoVE Journal

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

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 ...

연구

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 ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

성인 골격근의 개인 Myofibers 및 위성 세포의 분리와 문화

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

인간의 골격 근육에서 혈관 유래 다 능성 전구체의 분리

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

고해상도 호흡율 측정을위한 차동 원심 분리에 의한 골격근에서 본래 미토콘드리아의 분리

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

골격근 섬유아세포(Skeletal Muscle Fibroblast)에서 엑소좀의 분리 및 특성 분석(Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts)

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

질산염 및 아질산염 측정을 위한 쥐 골격근 균질액의 제조

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...