연구 KP에 NIH HL75503에 의해 지원되었다.

테네시 건강 과학 센터의 대학의 기관 애니멀 케어 및 사용위원회에 의해 승인 된 동물에서 수행 모든 실험이었다. 관류 쥐 폐 준비를위한 1. 관 <ol><li> 그림 1에서와 같이 압력 변환기 (P23XL)를 연결합니다. 혈액 관류에 대한 타이곤 튜브 (# 18)와 튜브 시스템을 준비하고 현미경 단계에 튜브를 배치합니다. </li><li> 37 ° C의 물을 욕조의 온도를 설정 </li><li> 폐의 입구를 연결하고 타이곤 튜브와 함께 일시적으로 출구. </li></ol><img alt="그림 1" fo:content-width="5in" src="/files/ftp_upload/51552/51552fig1highres.jpg" width="500" /> 그림 1. 혈액 관류 관. 회로도는 격리 된 폐 준비를 통해 혈액 순환에 사용되는 배관 설치를 보여줍니다. 또한 관련된 구성 요소가 있습니다 표시되는 통해 튜브 나의 라우팅. 심장과 폐의 회로도는 튜브 및 폐동맥 (PA)의 접속 위치를 표시하기 위해 포함, 심방 (LA) 정맥 (파란색 점선)에 남아 있습니다. 고립 된 쥐의 폐의 2. 준비 <ol><li> 케타민 (80 ~ 100 ㎎ / ㎏)과과 자일 라진 (5 ~ 10 ㎎ / ㎏)와, 쥐 (250-300그램 남성 흰쥐)를 마취. 마취의 수술 비행기를 확인한 후, 앙와위에서 동물을 배치합니다. </li><li> 기관 절개를 수행, 기관 캐 뉼러 (PE-90)를 삽입하고 수술 봉합사로 고정. </li><li> 심장 천자 (21 G 나비 바늘)에 의해 심장에 헤파린 (100 ~ 200 U)를 주입, 60 초 및에게서 피를 뽑다 혈액 (~ 12 ~ 15 ㎖의 혈액)을 기다립니다. </li><li> 두 캐 뉼러 (, 4 cm 길이, 타이곤 튜브 3 mm 직경의 한쪽 끝에 플레어)를 준비하고 식염수로 채우십시오. </li><li> 개흉술을 수행합니다. 우심실에 절개 (3 mm)를 확인하고 폐쪽으로 절개로 정맥의 플레어 끝을 밀어동맥. 봉합 수술과 폐 동맥에 정맥을 고정합니다. </li><li> 좌심실의 정점 (3 mm)를 절개하고 절개로 두 번째 정맥의 플레어 끝을 밀어 넣습니다. 좌심방으로 정맥을 안내합니다. 제대 테이프 (2 mm 폭)과 좌심실에 캐 뉼러를 고정합니다. </li><li> 모든 결합 조직을 해부하고, 첨부 된 캐 뉼러와 함께 폐와 심장을 제거합니다. 배양 접시에 폐와 심장을 놓습니다. 횡격막 표면 위에 있도록 폐의 위치를​​ 조정합니다. 세 캐 뉼러는 동일한 방향에 직면한다. </li><li> XY 방향으로 조작 할 수있는 단계에 폐 준비를 놓습니다. 현미경으로 오는 모든 단계는 튜브 또는 필요한 경우, 내장 맞춤 제작 단계를 유지하기 위해 수정 될 수있다. </li></ol> 고립 된 쥐의 폐의 3. 혈액 관류 <ol><li> 같은 부피의 알부민 용액 (5 %)로 exsanguinated 혈액을 혼합하고 저장을 추가 할 수 있습니다. 연동 펌프를 시작하고 흐름의 R 설정14 ㎖ / 분으로 먹었다. 혈액 튜브를 채울 수 있습니다. </li><li> 션트을 열어 둔 임시 폐 입구 출구 커넥터를 제거합니다. 각각 폐의 입구와 출구에 폐동맥과 왼쪽 심방 정맥을 연결합니다. 튜브에 기포가 없는지 확인합니다. </li><li> 제 3 압력 변환기 (P23XL)에 기관의 정맥을 연결합니다. 기관 캐뉼라를 통해 30 %의 산소와 폐를 팽창시키고 5cm의 H 2 O에 압력을 유지 </li><li> 폐 관류를 시작하는 클램프 션트을 닫습니다. 폐동맥 각각 10과 3 개 cm H 2 O ~에서 좌심방의 압력을 유지한다. </li></ol> 4. 미세 혈관 주입을위한 폐 준비 <ol><li> 폐 및 보안의 수준 위의 카테터의 확장 튜브의 후단을 올립니다. </li><li> ~ 길이 30cm PE90 튜브에 40 cm 길이 PE10 튜브의 약 30 센티미터 삽입하여 주입 카테터를 준비합니다. 바늘 (30 G)에서에 PE10 튜브의 노출 끝을 연결합니다주사기 (1 CC)에 블. </li><li> 확장 튜브의 제기 후 단부에 카테터 조합을 삽입합니다. 이 저항을 만날 때까지 좌심방을 통해 폐에 카테터 조합을 안내합니다. 이 저항을 충족 할 때까지, 폐에 더 혼자 PE10 카테터를 밀어 넣습니다. 카테터를 삽입 할 때 과도한 힘을 적용하면 폐를 손상되므로주의. </li><li> 링거액과 함께 1 CC의 주사기를 입력하고 주사기 펌프에 연결합니다. 10 μL / min으로 주입 속도를 설정합니다. </li><li> 주입 사이트는 폐의 나머지 부분에 비해 창백 될 것입니다. </li><li> 식염수로 폐를 적셔 발견 주입 사이트를 떠날 정도로 큰 구멍을 가진 플​​라스틱 포장으로 폐를 포함한다. </li><li> O-링 (맞춤 제작)의 바닥에 약간의 꼭지 그리스를 면봉 O-링의 바닥에 유리 커버 슬립 (# 1.5, 22mm 직경)를 배치합니다. 부드럽게 주입 사이트의 커버 슬립과 O-링을 배치합니다. 표준 테스트 튜브 홀리와 O-링을 고정데르. 최근 15를보고 커버 슬립 / O-링의 조합은, 그 아래 폐포 구조를 왜곡하지 않습니다. </li><li> O-링 위의 형광 현미경의 목적 (20X)를 놓고 폐 표면에 초점을 맞 춥니 다. </li></ol> 미세 혈관을 통해 5. 이미징 형광 덱스 트란 교통 <ol><li> 영상 FITC 개화에 대한 현미경을 준비합니다. </li><li> 이미지에 표시 할 영역을 선택하고, 이미지 수집 소프트웨어를 사용하여 1/minute의 속도로 이미지를 수집. </li><li> 주사기 펌프를 사용하여 FITC-덱스 트란 20 kD의 (0.5 ㎎ / ㎖)의 주입을 시작합니다. 혈관에 형광이 점차 증가하고 최대에 도달 할 것입니다. 60 분 동안 주입을 계속합니다. </li><li> 그런 내강 형광을 씻어 링거 주입으로 전환합니다. 10여 분 동안 주입을 유지한다. 워시 오프 동안 분당 1에서 이미지 수집을 계속합니다. </li></ol> 6. 이미지 분석 <ol><li> 이미지 수집 파일을 엽니 다. </li><li> 장소 지역이미지 프레임 내의 미세 혈관에 관심. </li><li> 이미지 파일을 프레임을 재생하고 모든 프레임에 대한 관심의 각 영역에서의 형광 강도를 기록한다. </li><li> 시간의 함수로서 각각의 관심 영역에 대한 형광 강도의 변화를 그린다. </li><li> 최대 형광 강도 및 washoff 10 분 후의 잔류 형광 강도를 계량화한다. </li><li> 관심의 지역과 관련된 미세 혈관의 투과성 인덱스를 얻기 위해 각 관심 영역에서 잔류 형광 강도에 최대의 비율을 계산합니다. </li></ol>

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저자가 공개하는 게 없다.

실시간 영상과 함께 고립 된 혈액 관류 폐 준비는 단일 폐의 미세 혈관의 투과성 변화의 측정을위한 간단한 도구를 제공합니다. 우리는 LPS의 주입에 대한 응답으로 투자율 변화를 정의하려면이 방법을 적용했다. 우리의 데이터는 명확하게 LPS 주입은 미세 혈관 투과성의 증가를 야기하는 것이 좋습니다. 또한, 데이터는 LPS에 의해 유도 투자율 변화 세정맥과 모세 혈관에서 모두 유사 하였다 것을 ?...

폐에 증가 된 미세 혈관의 투과성은 폐포 부종과 손상 가스 교환의 개발에 이르게 및 급성 폐 손상 (ALI) 1-3의 주요 특징입니다. 따라서, 혈관 투과성의 평가는 폐 손상과 제안 된 치료 적 개입의 효과의 범위를 정의하는 데 중요합니다. 혈액 무료 폐 습윤 건조 비율과 미세 혈관의 여과 계수와 같은 중량 분석 널리 투자율 4.5을 추정하는 방법을 사용합니다. 다른 방법은 폐 조직 6-8 방사성 형광 프로브의 유지를 정량화 있습니다. 그러나, 상기 방법은 투자율 데이터 해명 향해 폐 조직 샘플 postexperiment 가공을 필요로한다. 동물은 단일 치료 프로토콜에 대해서만 이용 될 수 있기 때문에 더욱, 큰 동물 번호는 완전한 연구를 위해 필요할 수도있다. 상기 방법의 일반적인 특징은 그들이 뜻 혈관 투과성을 결정한다는 것이다조직 샘플 내의 모든 혈관. 그러나, 잘 폐 마이크로 및 매크로 혈관 9 표현형 달라 확립된다. 따라서, 투과성 반응뿐만 아니라 9,10 다양한 용기 세그먼트 사이에서 이기종 수 있습니다. 따라서, 조직 샘플에있는 모든 폐 혈관의 평균 투자율을 정량화하는 것은 적절이 이질성을 반영하지 않을 수 있습니다. 절연 혈액 관류 폐 제제에서, 폐 표면에 혈관 직립 현미경 4,11,12 의해 시각화 될 수있다. 이 응답 (13)에서 임의의 이질성을 어드레싱, 따라서 단일 혈관의 특징 인 응답을 가능하게하고. 또, 미세 혈관의 형광 이미징을 이용하여 형광 분석법을 기반으로 통합 될 수있다. 또한, 좌심방 microcatheter 혈관 11,14으로 에이전트 및 형광 프로브를 전달하기 위해 사용될 수있다. microcatheter 작은 폐 영역에 배달 제한, 따라서 전주입 에이전트 및 형광에 지역 내에서만 혈관을 포즈. 이것은 동일한 폐 내의 체류 소 영역이 연구에 필요한 동물에서 전체적인 감소로 이어지는 별도의 실험에 사용되는 것을 허용한다. 실시간 영상은 혈관과 분리 된 폐 준비의 하나의 미세 혈관의 혈관 외 형광의 역동적 인 변화를 캡처 할 수 있습니다. 따라서, 이미지 필드 내의 각각의 미세 혈관에 대해, 형광체 및 washoff의 주입 동안 형광의 변화를 기록하고, 오프라인 (14)을 정량 할 수있다. 최대 및 잔류 혈관 형광의 값을 사용하여, 촬상 필드 내의 각 미세 혈관 투과성에 대한 인덱스가 결정될 수있다. 염증성 또는 유해한 제제에 응답 투자율 변화를 결정하기 위하여, 원하는 에이전트 제 투여 될 수 후 투자율 인덱스가 결정. 또, 이미지 필드는 주입에 의해 폐 영역 내의 아무 설정할 수microcatheter, 따라서 원하는 혈관 네트워크를 선택에서 높은 수준의 유연성을 가능하게한다. 따라서, 실시간으로 영상과 연계하여 고립 된 혈액 관류 폐 준비는 단일 폐의 미세 혈관의 투과성을 정량화 할 수있는 매력적인 실험 모델을 제공합니다.

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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tygon Tubing</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:297px;">#18</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pressure Transducer</td>
			<td style="width:108px;">Data Sciences International</td>
			<td style="width:119px;">P23XL</td>
			<td>Need quantity 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Butterfly Needle</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td align="right" style="width:119px;">450081</td>
			<td>21G</td>
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			<td height="21" style="height:21px;width:216px;">Peristaltic pump</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-90 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427421</td>
			<td>30 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-10 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427401</td>
			<td>40 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe Pump</td>
			<td style="width:108px;">Braintree Scientific</td>
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			<td height="21" style="height:21px;width:216px;">O-ring</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>Custom made with a 20 mm diamter hole&#160; and a handle&#160; to secure o-ring to holder</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Upright fluorescence microscope</td>
			<td style="width:108px;">Olympus America</td>
			<td style="width:119px;">BX61WI</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Image Acquisition Software</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC Dextran 20KD</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>0.5 mg/ml (A dextran of different molecular size can be selected, if trial experiments indicate its suitability based on the calculated permeability index values)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lipopolysaccharide</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>Serotype 0111:B4</td>
		</tr>
	</tbody>
</table>

quantifying single microvessel permeability in isolated blood-perfused rat lung preparation

고립 된 혈액 관류 폐 준비 널리 시각화하고 하나의 미세 혈관에 신호를 정의하는 데 사용됩니다. 실시간 이미징이 혼합물을 결합함으로써, 각각의 폐의 미세 혈관 투과성의 변화를 결정하기 위하여 가능해진다. 여기에서 우리는 쥐의 폐를 분리하고자가 혈액을 perfuse하는 단계를 설명합니다. 다음, 우리는 작은 폐 영역으로 microcatheter를 통해 형광 또는 대리인을 주입하는 단계를 설명합니다. 설명이 절차를 사용하여, 우리는 박테리아 리포 폴리 사카 라이드의 주입에 대한 응답으로 쥐의 폐의 미세 혈관의 투과성이 증가 결정. 데이터는 리포 폴리 사카 라이드가 모두 세정맥과 모세 혈관의 미세 혈관의 세그먼트에 걸쳐 유체 누출을 증가 것으로 나타났습니다. 따라서,이 방법은 가능한 혈관 세그먼트 구비 투과성 응답과 비교함으로써, 어떤 응답 이질성을 정의 할 수있다. 폐 투과성을 정의하기 위해 일반적으로 사용되는 방법으로는 폐 조직 샘플의 후 처리를 필요로하지만실시간 이미징의 사용은 본 방법에서 명백한 바와 같이 이러한 요구를 미연에 방지한다. 따라서, 실시간으로 영상과 함께 격리 된 폐 준비는 폐의 미세 혈관의 투과성을 결정하는 기존의 방법에 비해 여러 가지 장점을 제공합니다, 아직 개발하고 구현하는 간단한 방법입니다.

관류 배관 및 관련 장비에 연결된 절연 혈액 관류 폐 제조는도 2에 도시된다. 본 명세서에 기재된 방법은 랫트 ​​종으로 이용 될 수 있지만 설명을 위해, 우리는 스프 라그 돌리 래트를 사용 하였다. 좌심방 microcatheter 통해 주입은 폐의 작은 영역에 도달한다. 주입 영역 주입 유도 탈색 (도 3)에 의해 식별 될 수있다. 실시간 이미징 위치로 폐 제제는도 4에 도?...

Watch this Scientific Journal Video about Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation at JoVE.com

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

고립 된 혈액 관류 폐 준비는 폐 표면에 미세 혈관의 네트워크를 시각화하는 것이 가능합니다. 여기에서 우리는 실시간 형광 이미징을 사용하여 격리 된 폐의 단일 미세 혈관의 투과성을 정량화하는 방법을 설명합니다.

고립 된 혈액 관류 쥐 폐 준비에 단일 미세 혈관 투과성의 양을

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

quantifying-single-microvessel-permeability-isolated-blood-perfused

Research

JoVE Journal

Biology

9.4K 조회수.  The University of Tennessee Health Science Center.  고립 된 혈액 관류 폐 준비는 폐 표면에 미세 혈관의 네트워크를 시각화하는 것이 가능합니다. 여기에서 우리는 실시간 형광 이미징을 사용하여 격리 된 폐의 단일 미세 혈관의 투과성을 정량화하는 방법을 설명합니다. 

비디오: Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

gentleMACS Dissociator를 사용하여 마우스 폐 조직에서 단일 셀 Suspensions의 표준화 준비

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

연구

생물학

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

절연, 환기 및기구 마우스 폐의 특성화는 타악기 흐름 Perfused

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

예 생체내</em혈관 반응성 조사를위한> 절연 골격 Microvessel 준비

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

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 ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

삼투 물 투수 계수 (P 측정<sub&gt; F</sub&gt;) 구형 세포 : 예를 들어 식물 절연 원형질체

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

갓 격리 된 사구체 족 세포의 단일 채널 분석 및 칼슘 이미징

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusion 기술은 쥐 장간막에서 미세 혈관 투과성의 규정을 조사하기 위해

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146의 분리<sup&gt; +</sup쥐 폐에서&gt; 주민 폐 중간 엽 기질 세포

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

마우스입니다 관류 신장 기법

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

루트 엑수데이트에서 뿌리 균 화학 물질의 신속 한 식별에 대 한 향상 된 화학 분석

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...