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 약, 낮은 요구 사항이 고 일반적으로에서 얼룩 MMP 측정 셀 분리 할 미만 1.5 h 걸립니다.
몇 가지 중요 한 단계는 연구자 안정적이 고 만족 스러운 결과 얻을 하려는 경우 JC-1 시험을 수행할 때 과장 된 수 없습니다. 우선, JC 1 로드 셀 흐름 cytometer에 의해 수집 때까지 차가운 얼룩 버퍼에 메시지를 저장 합니다. 온도 MMP의 안정성에 상당한 영향 그리고 한 샘플의 값 실내 온도에 짧은 시간 후 많이 다릅니다. 또한, 예비 실험 권장 pretreating 시간 CCCP의 농도 결정 하 되도록 긍정적인 통제의 빨강/녹색 비율 ~ 0. 그것은 그 생성 시안 4-(trifluoromethoxy) phenylhydrazone (FCCP), 수 있습니다 또한 MMP를 제거 하 고 산화 인 산화를 억제, CCCP 대신 이며10긍정적인 제어 설정 하려면 사용할 수 있습니다.
JC-1 분석 결과 주요 한계는 다른 형광 JC-1와 결합 될 수 없습니다, 그리고 그것의 형광 방출 걸쳐 녹색, 노란색, 그리고 스펙트럼의 빨간색 파장의 부분. JC-1 분석 결과 mPTP의 시작 계정에 대 한 있는지 확인 하기 위해 calcein acetoxymethylester15,16 또는 심지어 유전자 조작17,18를 사용 하 여 하나 이상의 방법으로 결합 되어야 합니다 또한, MMP의 감소입니다. 이 때문에 ATP에 민감한 K+ 채널19,20 같은 다른 미토 콘 드리 아 숨 구멍의 오프닝 또한 미토 콘 드리 아의 도발은 이어질 수 있습니다.
JC-1 분석 결과 가장 적합 하다에 더 거친 "예/아니오" 미토 콘 드리 아 크게 인지 평가의 목표와 평가 편광 (예를 들어, apoptosis 관련 실험)8. 그것은 또한 MMP cardiomyocytes, 뉴런21, hepatocytes22, 신장 세포23 , 심지어 고립 된 미토 콘 드리 아24를 포함 하 여 다른 세포 유형의 다양 한 측정에 적용 되었습니다. 요약 하자면, 우리는 H/R. 후 HCMs에 MMP의 검출을 위한 빠르고, 간단 하 고 민감한 방법 설명

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 border="0" cellpadding="0" cellspacing="0" width="970">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Mitochondrial membrane potential assay kit with JC-1</td>
			<td style="width:307px;">Beyodtime, China</td>
			<td style="width:80px;">C2006</td>
			<td style="width:291px;">In the kit there are JC-1 stock solution (200&#215;), stock staining buffer (5&#215;) and CCCP(10mM)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tongxinluo ultrafine powder</td>
			<td>Shijiazhuang Yiling Pharmaceutical Co., China</td>
			<td>071201</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Annexin V-FITC/PI Kit</td>
			<td>Becton-Dickinson, USA</td>
			<td>556547</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM</td>
			<td>Life Technologies, Grand Island Biological Company, USA</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Human cardiac myocyte</td>
			<td>Promocell, Germany</td>
			<td>C-12810</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:293px;">Myocyte Growth Medium 
			(SupplementMix)</td>
			<td>Promocell, Germany</td>
			<td>C-39275</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">GENbox</td>
			<td>BioM&#233;rieux, Marcy l&#8217;Etoile, France</td>
			<td>96127</td>
			<td>2.5L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Catalyst (AnaeroPack)</td>
			<td>MITSUBISHI GAS CHEMICAL&#160;COMPANY, INC.&#160;, Japan</td>
			<td>&#160;C-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anaerobic indicator</td>
			<td>BioM&#233;rieux, Marcy l&#8217;Etoile, France</td>
			<td>96118</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow cytometer</td>
			<td>Becton-Dickinson, USA</td>
			<td>FACSAria 2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BD FACSDiva Software</td>
			<td>Becton-Dickinson, USA</td>
			<td></td>
			<td>Version8.0.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sample tube</td>
			<td>Corning science, USA</td>
			<td>352054</td>
			<td>12*75mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 ± 0.18% < 0.05). Tongxinluo, cardioprotective 효과와 중국 전통 의학 방해 HCMs H/R의 상태에서의 apoptosis (24.50 ± 1.13% p H/R 그룹에서 45.00 ± 2.13% < 0.05).
CCCP은 전자 전송 체인 전자 사업자의 정상적인 활동 동안 양성자 그라데이션 (MMP의 손실)을 분리 시에 의해 미토 콘 드리 아에서 산화 인 산화를 억제할 수 있는 protonophore 이다. 따라서, CCCP 치료 HCMs 긍정적인 통제로 설정 했다. 그림 4에서처럼 CCCP 치료 그룹에 빨강/녹색 비율 거의 제로 되었습니다. Apoptosis 분석 결과에서 결과와 일치, H/R-대우 그룹 표시 정상 그룹에 비해 훨씬 낮은 빨강/녹색 비율 (일반 그룹 p 에서 5.64 ± 0.21 대 0.69 ± 0.07 < 0.05)와 Tongxinluo 반전 같은 비율 변경 (2.92 ± 0.22 0.69 ± 0.07 pH/R 그룹에서 대 < 0.05).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57725/57725fig1.jpg"> 그림 1입니다. Hypoxia/reoxygenation (H/R) 모델의 설립. A. 재료; H/R 모델을 설정 하는 데 필요한 2. 항아리에 hypoxic 환경 확인. 혐 기성 표시기의 색 옅은 흰색 hypoxic 조건에 normoxic 환경에서 밝은 파란색에서 변경합니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57725/57725fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57725/57725fig2.jpg"> 그림 2입니다. Cytometry hypoxia/reoxygenation (H/R) 모델의 설립의 유효성 검사. 정상 그룹에 비해, 인간의 심장 myocytes의 apoptotic 속도 상당히 높은 H/R 그룹에 Tongxinluo 반전이 변화 하는 동안 (*p < 일반 대 0.05; # p < H/R 대 0.05; n = 3)11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57725/57725fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57725/57725fig3.jpg"> 그림 3입니다. 추가 분석에 대 한 FSC 점 음모 대 SSC에 세포 인구 (P1, 레드) 게이팅. 세포 인구의 무결성 측면 살포 (SSC)와 앞으로 분산형 (FSC)를 사용 하 여 평가 이다. 빛 감소 (외부 게이트 P1, 블랙), 세포 파편 비 산, 제외 됩니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57725/57725fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57725/57725fig4.jpg"> 그림 4입니다. 미토 콘 드 리아 멤브레인 인간의 심장 myocytes 잠재력의 평가. 정상 그룹에 비해, H/R 그룹 표시 빨강/녹색 비율을 크게 낮춰 Tongxinluo이이 추세 반전 하는 동안 (빨강: 셀 아무 형광; 빨간 형광; 블루, 셀 녹색 형광으로 보라색, 셀입니다. 음성 제어, 빈 고 얼룩이 지기; 긍정적인 통제, 셀 CCCP;로 치료 p < 0.05 정상; 대 #p < 0.05 H/R; 대 n = 3)11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57725/57725fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>

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

Nanyang Technological University

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

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory rate, salivation, perspiration and renal function. The ANS has two main branches: the sympathetic nervous system, preparing the human body for action in times of danger and stress, and the parasympathetic nervous system, which regulates the resting state of the body.
ANS activity can be measured invasively, for instance by radiotracer techniques or microelectrode recording from superficial nerves, or it can be measured non-invasively by using changes in an organ's response as a proxy for changes in ANS activity, for instance of the sweat glands or the heart. Invasive measurements have the highest validity but are very poorly feasible in large scale samples where non-invasive measures are the preferred approach. Autonomic effects on the heart can be reliably quantified by the recording of the electrocardiogram (ECG) in combination with the impedance cardiogram (ICG), which reflects the changes in thorax impedance in response to respiration and the ejection of blood from the ventricle into the aorta. From the respiration and ECG signals, respiratory sinus arrhythmia can be extracted as a measure of cardiac parasympathetic control. From the ECG and the left ventricular ejection signals, the preejection period can be extracted as a measure of cardiac sympathetic control. ECG and ICG recording is mostly done in laboratory settings. However, having the subjects report to a laboratory greatly reduces ecological validity, is not always doable in large scale epidemiological studies, and can be intimidating for young children. An ambulatory device for ECG and ICG simultaneously resolves these three problems.
Here, we present a study design for a minimally invasive and rapid assessment of cardiac autonomic control in children, using a validated ambulatory device 1-5, the VU University Ambulatory Monitoring System (VU-AMS, Amsterdam, the Netherlands,<a href="http://www.vu-ams.nl" target="_blank"> www.vu-ams.nl</a>).

Measuring Cardiac Autonomic Nervous System ANS Activity in Children

Measurement of autonomic nervous system activity usually confines the researcher and participant to the laboratory, which may provide an intimidating environment to children. The VU University Ambulatory Monitoring System (VU-AMS) device can record cardiac autonomic control in any setting. The VU-AMS proved very amenable to testing in children.

아이들의 측정 심장 자율 신경계 (ANS) 활동

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory ...

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Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

Imaging- 및 유동 세포 계측법 기반의 셀 위치의 분석 및 3D 흑색 종 회전 타원체의 세포주기

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

인-31 자기 공명 분광학 : 측정을위한 도구<em&gt; 생체</em인간의 골격 근육에&gt; 미토콘드리아 산화 적 인산화 능력

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

심장 수술 후 혈청 샘플의 영향을 건강한 자원 봉사자와 그들의 철새 잠재력에서 내피 전구 세포의 분리

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function are monitored by the change in voltage across the nasal epithelium after the superfusion of solutions that modify ion channel activity. This is enabled by the measurement of the potential difference between the subcutaneous compartment and the airway epithelium in the nostril, utilizing a catheter in contact with the inferior nasal turbinate.
The test allows the measurement of the stable baseline voltage and the successive net voltage changes after perfusion of 100 &#181;M amiloride, an inhibitor of Na+ reabsorption in Ringer&#39;s solution; a chloride-free solution containing amiloride to drive chloride secretion and 10 &#181;M isoproterenol in a chloride-free solution with amiloride to stimulate the cyclic adenosine monophosphate (cAMP)-dependent chloride conductance related to CFTR.
This technique has the advantage of demonstrating the electrophysiological properties of two key components establishing the hydration of the airway surface liquid of the respiratory epithelium, ENaC, and CFTR. Therefore, it is a useful research tool for phase 2 and proof of concept trials of agents that target CFTR and ENaC activity for the treatment of cystic fibrosis (CF) lung disease. It is also a key follow-up procedure to establish CFTR dysfunction when genetic testing and sweat testing are equivocal. Unlike sweat chloride, the test is relatively more time consuming and costly. It also requires operator training and expertise to conduct the test effectively. Inter- and intra-subject variability has been reported in this technique especially in young or uncooperative subjects. To assist with this concern, interpretation has been improved through a recently validated algorithm.

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure.

코 막 Transepithelial 잠재력 차이 (NPD)의 표준화 된 측정

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

기니 돼지 모델에서 심근 손상 후 심장 근육 패치 hiPSC 파생의 이식

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

성인 마우스에서 심방 근세포의 격리

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

인간의 혈류를 모방하는 흐름 루프 모델에서 혈액 접촉 임플란트의 혈적합성 테스트

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

인간 심장 근세포 모델에서 저체온증-사전 조절 에 따른 심근 보호의 체외 평가

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...