이 연구는 국가 핵심 연구 및 개발 프로그램의 중국 (No. 2017YFC1700503)에서 교부 금에 의해 지원 되었다 국가 기본 연구 프로그램 (973 프로그램) 중국 (No.2012CB518602), 국립 자연 과학 재단의 중국 (No. 81370223 그리고 번호 81573957), 북경 연합 대학 (2016-1002-01-02)의 대학원생 혁신적인 연구 재단.

1입니다. 시 약 및 솔루션의 준비
<ol><li>제조업체의 지침에 따라 Myocyte 성장 매체의 500 ml Myocyte 성장 매체 보충 믹스의 25 mL를 추가 하 여 인간의 심장 myocytes (HCMs) 완전 한 매체를 준비 합니다. 4 ° C에서 37 ° c를 사용 하기 전에 따뜻한이.</li>
	<li>Tongxinluo (광장) 솔루션 준비 혈 청/포도 당-무료 광장 ultrafine 분말을 용 해 하 여 Dulbecco의이 글의 중간 (DMEM)를 수정 하 고 (자세한 내용은 참조11)에 대 한 DMEM을 추가 하 여 400 µ g/ml 광장의 농도 조정. 4 ° C에서 37 ° c를 사용 하기 전에 따뜻한이.</li>
	<li>JC-1 재고 솔루션 (200 x), 초순, 4 mL 및 재고 버퍼 (5 배) 15 mL 원심 분리기 튜브 어둠 속에 얼룩의 1 mL의 25 µ L을 추가 하 여 제 1 작업 솔루션의 5 mL을 준비 합니다. 여러 번 왔다 갔다 혼합 플라스틱 고 37 ° C를 사용 하기 전에 따뜻한.</li>
	<li>30 mL 얼룩 버퍼 초순의 24 mL 50 mL 원심 분리기 튜브에 버퍼 (5 배) 얼룩 재고의 6 mL에 추가 하 여 작업을 준비 합니다. 얼음 처럼 차가운이 솔루션을 사용 합니다.</li>
</ol>2입니다. 저 산소 증/Reoxygenation 모델의 설립
<ol><li>호치민시 6 잘 플레이트에 전체 매체와 당 판 1.0 x 105 셀. 37 ° c.에 5% CO2 와 95% 공기를 포함 하는 셀 인큐베이터에 약 70%의 confluency에 도달 성장</li>
	<li>씻어 HCMs 인산 염 버퍼 식 염 수 (PBS). 다른 치료에 그들을 노출 (400 µ g/mL 광장 안) 2 mL 혈 청/포도 당 자유로운 DMEM 37 ° c.에 5% CO2 와 95% 공기를 포함 하는 셀 인큐베이터에서 hypoxia 이전 30 분에</li>
	<li>무료 산소 청소 18 h (그림 1A), 저 산소 증 및 중간11,12, 에서 산소 긴장을 측정 하는 혐 기성 표시기를 유도 하는 밀폐 및 hypoxic 항아리 (2.5 L) 촉매 장착에 HCMs 품 어 13, 5% CO2 와 95% 공기에서 37 ° c.를 포함 하는 셀 인큐베이터에서 참고: 그것은 약 1.5 h 항아리에 무료 산소 청소를 촉매에 대 한 걸립니다. 따라서, 단지 열 전에 HCMs 19.5 h 항아리에 보관 됩니다. 혐 기성 표시기의 색 옅은 흰색 hypoxic 조건 (그림 1B)에 normoxic 환경에서 밝은 파란색에서 변경합니다.</li>
	<li>6 잘 플레이트 인큐베이터 2 h 5% CO2 와 95% 공기를 포함 하는 37 ° C에서 설정으로 이동 하 여 HCMs reoxygenate.</li>
</ol>3입니다. Cytometry HCMs의 준비
<ol><li>37 ° c는 0.25% trypsin ethylenediaminetetraacetic 산 (EDTA) 솔루션 및 PBS를 따뜻한</li>
	<li>각 우물에서 매체를 발음, 1 mL의 PBS로 세포를 씻어 하 고 우물의 벽을 따라 0.25 %trypsin 0.5 mL를 추가 합니다.</li>
	<li>모든 HCMs 거의 분리 (약 1 분) 때까지 37 ° C에서 품 어.</li>
	<li>중화는 트립 신을 우물에 DMEM 완전 한 매체 (10% 태아 둔감 한 혈 청으로 DMEM 매체)의 1 mL를 추가 합니다. 15 mL 원심 분리기 튜브에 세포 현 탁 액을 전송 하 고 HCMs centrifuging 실내 온도에 3 분 동안 500 x g에 의해 작은.</li>
	<li>신중 하 게는 펠 렛을 방해 하지 않고는 상쾌한 발음.</li>
	<li>각 튜브를 0.5 mL 호치민시 완전 한 매체의 작품 및 JC-1 작업 솔루션의 0.5 mL를 추가 합니다. 아래로 pipetting으로 HCMs resuspend.</li>
	<li>전체 튜브 형광 표시기의 표백 방지 하기 위해 알루미늄 호 일을 커버 하 고 20 분 동안 HCMs (5% CO2 와 95% 공기를 포함 하는 37 ° C)에서 정상적인 조건에서 품 어.</li>
	<li>펠 릿 HCMs centrifuging 500 x g 4 ° c.에 3 분에 의해</li>
	<li>얼음 처럼 차가운 JC-1 얼룩 버퍼의 2 개 mL 두번 HCMs 린스 합니다.</li>
	<li>샘플 튜브 (12 x 75 mm)에 얼음 처럼 차가운 얼룩 버퍼의 300 µ L의 최종 볼륨에서 HCMs resuspend 고 30 분 이내 분석에 대 한 셀을 사용 합니다. 참고: 카보닐기 시안 m chlorophenyl hydrazone (CCCP), 호흡 전자 수송 체인의 강력한 억제제를 사용 하 여 긍정적인 통제에 대 한 20 µ M의 농도에서 1 h HCMs 치료.</li>
</ol>4. 교류 Cytometer 설치
<ol><li>교류 cytometer 시작 하 고 소프트웨어에 연결 되어 있는지 확인.</li>
	<li>응용 시작 수행, 스트림을 시작 하 고는 breakoff를 설정.</li>
	<li>교류 cytometry 소프트웨어에서 두 점 플롯 윈도우를 엽니다.<ol>
<li>선택 앞으로 각각 그들의 크기 및 그들의 단위 셀의 특성을 표현 하기 위해 첫 번째 점 플롯에서 Y 축에 X 축에 빛 (FSC)과 측면 흩어져 빛 (SSC) 흩어져.</li>
		<li>X 축 눈금을 사용 하 여 셀에 JC 1 염료의 형광 강도 측정 하는 데 사용 되는 두 번째 점 플롯에 대 한 PE (575 ± 26 nm) 탐지 채널 Y 축과 FITC (530 ± 30 nm)에 대 한 선택 합니다.</li>
	</ol>
</li>
</ol>5. 흐름 Cytometry 측정 및 데이터 분석
<ol><li>튜브 밖으로 팔을 지원 하 고는 빈 (HCMs 하지 염색 하는 JC-1) 위치에 있도록 언로드 클릭 그것에 튜브 샘플. 튜브 지원 팔 작업 위치로 반환 부하 를 클릭 합니다.</li>
	<li>레코드 빈 샘플 튜브에서 현 탁 액에서 입자를 수집 하 고 다음 셀 인구 (P1) 첫 도트 작 (그림 3)에서 추가 분석을 위해 게이트를 클릭 합니다.</li>
	<li>다른 샘플 튜브에서 HCMs를 수집 하 고 셀 점 두 번째 점 플롯의 중심 지역에 위치 하 고 있는지 확인을 형광 채널의 전압을 조정 하 여 측정을 최적화.</li>
	<li>각 샘플 튜브의 통계를 표시 하 고 녹색 형광 강도의 빨간 형광 강도의 비율을 분할 하 여 각 샘플의 MMPs 계산.</li>
</ol>

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저자는 공개 없다.

여기, 선물이 JC-1 분석 결과 의해 H/R. 검색에 노출 되 고 후 셀의 MMP를 평가 하기 위해 JC 1 염료를 사용 하는 프로토콜, 세포의 MMP는 미토 콘 드리 아의 크기, 모양, 및 형광 단일 구성 요소에 영향을 미칠 수 밀도 같은 요인의 독립 14신호. 따라서, JC-1 분석 결과의 결과 상대적으로 안정적 이다. 게다가, 그것은 편리 하 고 JC-1 시험 수행에 시간 절약입니다. 이 분석 결과 재료 및 시 ?...

관상 동맥 심장 질환 죽음은 전세계의 주요 원인입니다. 치료의 선택의 허 혈 성 상해를 줄이고 ST 세그먼트를 가진 환자에서 경색 크기 제한 상승 심근 경색 주 경 피 적인 관상 동맥 내정간섭 (PCI)1를 통해 신속 하 고 효과적인 심근 reperfusion는 2. 그러나, reperfusion 최종 경색 크기3의 30%를 위한 계정을 수 있습니다 추가 손상이 발생 합니다. 그것은 보편적으로 미토 콘 드 리아 침투성 전환 기 공 (mPTP)가 뿐만 아니라 국 소 빈 혈 또는 reperfusion 동안 미토 콘 드 리아 손상과 세포 죽음에 중앙 인정 (I / R), cardioprotective 신호4 의 수렴 대상 이기도 하지만 , 5. 우리 mPTP 오프닝 5 ', 5를 사용 하 여 감지 mPTP 오프닝 안 미토 콘 드리 아 막 잠재적인 (MMP)4의 도발은 대해가지고 것, 6, 6 '-Tetrachloro-1, 1, 3, 3 '-tetraethyl-imidacarbocyanineiodide (JC-1) 분석 결과.
JC-1 분석 결과 cytofluorimetric 메서드를 질적, 양적, 그리고 단일 미토 콘 드리 아6의 수준에서 MMP를 분석 하 여 추가 검증 되었습니다. JC-1 오렌지 색깔된 방출에 (590 ± 17.5 nm) 매트릭스에서 정상적인 MMP;와 미토 콘 드리 아의 빨간색 저조한, 집계 된 형태로 존재 MMP의 손실, JC-1 530 ± 15 nm의 방출 함께 녹색 형광을 생성 하는 단위체 양식으로 변환 됩니다. 따라서, 빨강/녹색 형광 강렬 비율 감소 국 소 빈 혈 또는 reperfusion 같은 조건에서 MMP의 감소를 나타낼 수 있습니다 (I / R).
JC-1, 이외에 MMP Rhodamine 123 등 3, 3 '-dihexyloxadicarbocyanine 요오드 화물 [DiOC6(3)] 막 투과성 질 성 양이온으로 공부도 했다. 그러나, 이러한 두 프로브에 비해, JC 1 MMP를 분석 하기 위한 더욱 안정적입니다. Rhodamine 123 (특히 냉각 모드7,8)에서 상대적으로 빈약한 감도 있으며 가난한 특이성. Rhodamine 123에에서 변화 때로는 작습니다 그래서 그것은 관찰/감지 하 연구원 또는 장비에 대 한 하드. 게다가, 단일 셀에서 rhodamine 123에 대 한 다른 mitochondrion 바인딩 사이트 하 고 그래서 그것이 다른 형광 방출9있을 수 있습니다 키를 누릅니다. DiOC6(3) MMP 감지에 대 한 권장 하지 않습니다도 플라즈마 막10의 도발은에 민감하게 반응 하는 그것.
따라서, 여기에 우리가 hypoxia/reoxygenation 또는 보호 에이전트 없이 노출 되 고 후 HCMs MMP를 평가 하기 위해 JC-1 분석 결과 사용 합니다.

<table><tbody><tr><td>Mitochondrial membrane potential assay kit with JC-1</td><td>Beyodtime, China</td><td>C2006</td><td>In the kit there are JC-1 stock solution (200&amp;times;), stock staining buffer (5&amp;times;) and CCCP(10mM)</td></tr><tr><td>Tongxinluo ultrafine powder</td><td>Shijiazhuang Yiling Pharmaceutical Co., China</td><td>071201</td><td></td></tr><tr><td>Annexin V-FITC/PI Kit</td><td>Becton-Dickinson, USA</td><td>556547</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies, Grand Island Biological Company, USA</td><td>11966-025</td><td></td></tr><tr><td>Human cardiac myocyte</td><td>Promocell, Germany</td><td>C-12810</td><td></td></tr><tr><td>Myocyte Growth Medium&lt;br/&gt; (SupplementMix)</td><td>Promocell, Germany</td><td>C-39275</td><td></td></tr><tr><td>Myocyte Growth Medium (Ready-to-use)</td><td>Promocell, Germany</td><td>C-22070</td><td>used with Myocyte Growth Medium SupplementMix</td></tr><tr><td>GENbox</td><td>BioM&amp;eacute;rieux, Marcy l&amp;rsquo;Etoile, France</td><td>96127</td><td>2.5L</td></tr><tr><td>Catalyst (AnaeroPack)</td><td>MITSUBISHI GAS CHEMICAL&amp;nbsp;COMPANY, INC.&amp;nbsp;, Japan</td><td>&amp;nbsp;C-1</td><td></td></tr><tr><td>Anaerobic indicator</td><td>BioM&amp;eacute;rieux, Marcy l&amp;rsquo;Etoile, France</td><td>96118</td><td></td></tr><tr><td>Flow cytometer</td><td>Becton-Dickinson, USA</td><td>FACSAria 2</td><td></td></tr><tr><td>BD FACSDiva Software</td><td>Becton-Dickinson, USA</td><td></td><td>Version8.0.1</td></tr><tr><td>Sample tube</td><td>Corning science, USA</td><td>352054</td><td>12*75mm</td></tr><tr><td>PBS</td><td>Hyclone, USA</td><td>SH30256.01</td><td></td></tr></tbody></table>

a flow cytometry-based assay for measuring mitochondrial membrane potential in cardiac myocytes after hypoxia/reoxygenation

관상동맥 폐색된의 신속 하 고 효율적인 reperfusion ST 세그먼트를 가진 환자에서 심근 경색 크기를 감소에 대 한 최선의 전략 상승 심근 경색 이다. 그러나, reperfusion 라기보다 발생할 수 있습니다 추가 cardiomyocyte 죽음, reperfusion 상해로 알려진 현상. (MMP), 미토 콘 드리 아 막 잠재력의 감소와 미토 콘 드리 아의 도발은 미토 콘 드 리아 침투성 전환 기 공 (mPTP)의 오프닝 reperfusion 상해의 마지막 단계로 보편적으로 인식 되 고에 대 한 책임은 미토 콘 드리 아와 cardiomyocyte 죽음. JC-1 MMP의 값에 따라 미토 콘 드리 아에서 축적 되는 질 성 양이온 염료 이다. MMP, JC-1는 미토 콘 드리 아에서 축적 하는 더 높은. 미토 콘 드리 아에서 JC-1의 증가 금액에서 녹색 형광 방출 변화에 의해 반영 될 수 있다 (~ 530 nm) 빨강 (~ 590 nm). 따라서, 빨강/녹색 형광 강렬 비율의 감소는 미토 콘 드리 아의 도발은 나타낼 수 있습니다. 여기, 우리 걸릴 JC-1 MMP, 측정을 활용 또는 산소/reoxygenation, cytometry에 의해 감지 후 인간의 심장 myocytes에서 mPTP의 오프닝.

MMP의 변화를 평가 하기 위해 JC-1 분석 결과 수행 하기 전에 실험 실시 성공적으로 조건을 확인 하는 연구자에 의해 설정 것이 좋습니다. 정상 그룹에 비해 교류 cytometry 결과 (그림 2)와 같이 산소/reoxygenation (H/R)는 크게 HCMs (Annexin V + /PI±)의 apoptosis 유도 우리 나의 세포 기반 모델을 설립 했다 나타내는 / R (45.00 ± 2.13 %vs. 11.50 p 일반 그룹에서 ± 0.1...

Watch this Scientific Journal Video about A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation at JoVE.com

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

여기, 선물이 hypoxia/reoxygenation 또는 보호 에이전트 없이 노출 되 고 후 세포의 미토 콘 드리 아 막 잠재력을 평가 하기 위해 JC 1 염료를 사용 하는 프로토콜.

Hypoxia/Reoxygenation 후 심장 Myocytes에서 미토 콘 드리 아 막 잠재력을 측정 하기 위한 교류 Cytometry 기반 분석

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

a-flow-cytometry-based-assay-for-measuring-mitochondrial-membrane

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

14.1K 조회수.  Chinese Academy of Medical Sciences and Peking Union Medical College. 여기, 선물이 hypoxia/reoxygenation 또는 보호 에이전트 없이 노출 되 고 후 세포의 미토 콘 드리 아 막 잠재력을 평가 하기 위해 JC 1 염료를 사용 하는 프로토콜.

비디오: A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

DHODH 및 SDH 활성을 측정하기 위한 안정 동위원소 추적 및 LC-MS 분석

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

저자 스포트라이트: 포유류의 미토콘드리아 기능을 측정하기 위한 산소 독립적 분석  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

포유류의 미토콘드리아 기능을 측정하기 위한 산소 비의존적 분석

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

연구

생화학

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

사포닌 - permeabilized 심장 섬유에 Respirometric 산화 인산화 평가

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

생물학

Mitochondrial Functional Assessment in Brush Lizard Liver Mitochondria

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of ...

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 ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

세포 외 유출 분석기를 사용 하 여 기본 교양된 마우스 신생아 Cardiomyocytes에 산소 소비 속도 분석

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

의학

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

고립 된 미토콘드리아의 유동 세포 계측법에 의해 외부 막 단백질의 면역

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

형광 현미경으로 살아있는 세포의 잠재적 인 미토콘드리아의 칼슘과 미토콘드리아 막의 동시 측정

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

에플로프 펌프 억제를 통해 조혈 줄기 및 전조 세포에서 미토콘드리아 막 전위성 유동 세포 측정 평가의 정확도 향상

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Hypoxia/Reoxygenation 후 심장 Myocytes에서 미토 콘 드리 아 막 잠재력을 측정 하기 위한 교류 Cytometry 기반 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

포유류의 미토콘드리아 기능을 측정하기 위한 산소 비의존적 분석

포유류의 미토콘드리아 기능을 측정하기 위한 산소 비의존적 분석

포유류의 미토콘드리아 기능을 측정하기 위한 산소 비의존적 분석

사포닌 - permeabilized 심장 섬유에 Respirometric 산화 인산화 평가

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

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

세포 외 유출 분석기를 사용 하 여 기본 교양된 마우스 신생아 Cardiomyocytes에 산소 소비 속도 분석

고립 된 미토콘드리아의 유동 세포 계측법에 의해 외부 막 단백질의 면역

형광 현미경으로 살아있는 세포의 잠재적 인 미토콘드리아의 칼슘과 미토콘드리아 막의 동시 측정

에플로프 펌프 억제를 통해 조혈 줄기 및 전조 세포에서 미토콘드리아 막 전위성 유동 세포 측정 평가의 정확도 향상