研究は、KPにNIH HL75503によってサポートされていました。

テネシー大学健康科学センターの施設内動物管理使用委員会によって承認された動物で行われ、すべての実験であった。 灌流はラット肺の準備のための1。チューブ<ol><li> 図1に示すように、圧力変換器（P23XL）を接続します。血液灌流用タイゴンチューブ（＃18）と配管システムを準備し、顕微鏡ステージ上にチューブを置きます。 </li><li> 37℃の水浴温度を設定する</li><li>肺の入口および出口はタイゴンチューブを一時的に接続する。 </li></ol><img alt="図1" fo:content-width="5in" src="/files/ftp_upload/51552/51552fig1highres.jpg" width="500" /> 図1。血液灌流チューブ 。回路図は、孤立した肺の準備を通して血液を循環させるために使用される管のセットアップを示しています。また、示されたが、部品を通る配管I関連しているSがルーティングされた。心臓と肺の概略管および肺動脈（PA）、および左心房（LA）、カニューレ（青色破線）との接続部位を示すために含まれている。 ラット摘出肺の2。準備<ol><li>ケタミン（80〜100 mg / kg）を持つとキシラジン（5月10日mg / kg）を持つ、ラット（250〜300グラムのオスのSDラット）を麻酔。麻酔の外科的平面性を確保した後、仰臥位で動物を配置します。 </li><li>気管切開を行い、気管カニューレ（PE-90）を挿入し、外科用縫合糸で固定します。 </li><li>心臓穿刺（21のG翼状針）で心にヘパリン（100〜200 U）を注入、60秒、放血させる血液（〜12〜15ミリリットルの血液）を待つ。 </li><li> 2カニューレ（;長さ4cm、タイゴンチューブ、直径3mm一端にフレア）を準備し、生理食塩水で満たします。 </li><li>開胸を行います。右心室に切開（3ミリメートル）を作成し、肺に向かって切開部にカニューレのフレア端部をスライドさせ動脈。手術用縫合糸で肺動脈にカニューレを固定します。 </li><li> （3ミリメートル）左心室の頂点を切開し、切開部に第二のカニューレのフレア端部をスライドさせます。左心房にカニューレをガイド。臍テープ（2ミリメートル幅）を左心室にカニューレを固定します。 </li><li>任意の結合組織を解剖し、付属のカニューレと一緒に肺と心臓を取り出します。ペトリ皿に、肺や心臓を配置します。横隔膜面が上になるように、肺の位置を変更。 3カニューレは、同じ方向を向くようにします。 </li><li> XY方向に操作することができ、ステージ上の肺の準備を置く。顕微鏡に付属しているいずれかの段階では、チューブや、必要に応じて、構築されたカスタムメイドの段階を保持するように変更することができます。 </li></ol>ラット摘出肺の3。血液灌流<ol><li>同体積のアルブミン（5％）溶液を用いて放血血液を混ぜてリザーバに追加します。蠕動ポンプを起動し、フローRを設定14ミリリットル/分に食べました。血液がチューブを埋めることができます。 </li><li>シャント開いて、一時的な肺の出入口コネクタを外します。それぞれ、肺の入口と出口に、肺動脈と左心房のカニューレを取り付けます。配管内に気泡がないことを確認してください。 </li><li>第3の圧力トランスデューサー（P23XL）に気管カニューレを接続します。気管カニューレを介して30％の酸素で肺を膨張させ5cmのH 2 Oの圧力を維持する</li><li>肺灌流を開始するクランプでシャントを閉じます。 〜10で、肺動脈と左心房の圧力を維持し、3センチメートルH 2 Oであった。 </li></ol> 4。微小血管注入のための肺の準備<ol><li>肺で安全のレベルを超えてカテーテル用延長チューブの後端を持ち上げます。 </li><li>約30センチPE90チューブに長さ40cmのPE10チューブを約30cmを挿入して注入カテーテルを準備します。針（30 G）のATにPE10チューブの露出端を接続注射器（1 CC）に取り付けられ。 </li><li>延長チューブの隆起後端にカテーテルの組み合わせを挿入します。それは抵抗を受けるまで、左心房を通って肺にカテーテルの組み合わせを導く。それは抵抗を受けるまで、肺の中にさらに単独のPE10カテーテルを押してください。カテーテルを挿入しながら、無理な力を適用することは、肺を傷つけることに注意してください。 </li><li>リンゲル液で1ccの注射器を満たし、シリンジポンプに取り付けます。 10μL/分に注入速度を設定します。 </li><li>注入部位は、肺の他の部分に比べて薄いとなります。 </li><li>生理食塩水で肺を湿らし、明らかに注入部位を残すのに十分な大きさの穴をラップで肺をカバーしています。 </li><li> Oリング（オーダーメイド）の下にいくつかのストップコックグリースを綿棒＆Oリングの底にガラスカバースリップ（＃1.5、直径22mm）を配置します。優しく注入部位でのカバーガラスでOリングを配置。標準試験管HOLにOリングを固定しますデア。最近、15を報告したようにカバースリップ/ Oリングの組み合わせは、その下歯槽構造を歪曲していません。 </li><li>位置Oリング、上記蛍光顕微鏡の対物レンズ（20X）および肺表面に焦点を当てる。 </li></ol>微小血管スルー5。イメージング蛍光デキストラントランジット<ol><li> FITCの蛍光を画像化するための顕微鏡を準備します。 </li><li>撮像すべき領域を選択し、画像取得ソフトウェアを用いて1/minuteの速度で画像を取得する。 </li><li>シリンジポンプを使用してFITC-デキストラン20kDの（0.5 mg / ml）での注入を開始する。血管内の蛍光が徐々に増加し、最大値に達する。 60分間注入を続ける。 </li><li>その後、管腔の蛍光を洗い流しリンゲル輸液に切り替える。 10分かけて点滴用を維持する。洗い流し時の限界回転で画像収集を続ける。 </li></ol> 6。画像解析<ol><li>画像取得ファイルを開きます。 </li><li>場所領域画像フレーム内の微小血管の上の関心の。 </li><li>イメージファイルフレームごとに再生すると、すべてのフレームのための関心の各領域での蛍光強度を記録。 </li><li>時間の関数としての関心領域毎の蛍光強度の変化をプロットする。 </li><li>ウォッシュオフの10分後、最大蛍光強度と残留蛍光強度を定量化する。 </li><li>関心のその領域に関連する微小血管の透過性の指標を得るために、関心の各領域での残留蛍光強度を最大の比率を計算します。 </li></ol>

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著者らは、開示することは何もありません。

リアルタイムイメージングと相まって孤立血液潅流肺標本は、単一の肺の微小血管内の透過性の変化を測定するためのシンプルなツールを提供しています。我々は、LPSの注入に応じて透過性の変化を定義するには、この方法を適用した。我々のデータは明らかに、LPS注入は、微小血管透過性の増加を引き起こしたことを示唆している。また、データも、LPSによって誘導された透過性の変化は?...

肺における微小血管透過性の増加は、浮腫および肺胞の妥協ガス交換の開発につながり、急性肺損傷（ALI）1-3の主要な特徴である。従って、血管透過性の推定値は、肺損傷および提案された治療的介入の有効性の範囲を定義する上で重要である。このような血中遊離肺のウェット対ドライ比と微小血管ろ過係数として重量分析は、広く浸透性4,5を推定するための方法を使用します。他の方法は、肺組織中の6-8放射性または蛍光プローブの保持を定量することが挙げられる。しかしながら、上記の方法は、透過性データを解明に向けて肺組織サンプルのpostexperiment処理を必要とする。一匹の動物は、単一の治療プロトコルに使用することができるので、大型動物番号は完全な研究のために必要とされ得る。上記の方法の共通の特徴は、彼らが平均血管透過性を決定するということです組織サンプル内の全ての血管。しかし、周知の肺ミクロおよびマクロ血管が表現型的に異なっている9ことが確立されている。このため、透過性の応答は、同様に9,10の様々な血管セグメントの中で異質かもしれません。このように、組織サンプル中のすべての肺血管の平均透過性を定量化することは十分にこの不均一性を反映しない場合があります。 単離された血液灌流肺製剤において、肺表面上の血管が正立顕微鏡4,11,12によって可視化することができる。これは、単一の容器内の応答を特徴づけるため、応答13内の任意の不均一性に対処することができます。また、微小血管の蛍光イメージングを利用して、蛍光ベースのアッセイは、組み込むことができる。また、左心房のマイクロカテーテルは、血管11,14内剤および蛍光プローブを送達するために使用することができる。マイクロカテーテルは、小さな肺領域への配信を制限し、このようにEX注入された薬剤と蛍光団にリージョン内でのみ血管を装った。これは、同じ肺内の複数の小領域は、研究に必要な動物の全体的な減少をもたらす、別々の実験のために使用することができる。 リアルタイムイメージングは​​、孤立した肺の準備の単一微小血管の血管および血管外蛍光の動的な変更の取得を可能にします。したがって、画像フィールド内の各微小血管のために、フルオロフォアおよびウォッシュオフの注入の間蛍光の変化を記録し、オフライン14を定量することができる。最大残留血管の蛍光値を用いて、撮像視野内の各微小血管の透過性指数を決定することができる。炎症性または有害な薬剤に応じて、透過性の変化を決定するために、所望の薬剤を最初に投与することができ、その後の透過性指数を決定した。また、画像フィールドには、注入によって肺の領域内のどこにでも設定することができるマイクロカテーテル、所望の血管網を選択する際の高度な柔軟性を可能にする。このように、リアルタイムで画像化と並行して孤立した血液灌流肺の準備は、単一の肺の微小血管内の透過性を定量化するための魅力的な実験モデルを提供しています。

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peristaltic pump</td>
			<td style="width:108px;">Cole Parmer</td>
			<td style="width:119px;">Masterflex L/S</td>
			<td></td>
		</tr>
		<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>
			<td style="width:119px;">BS8000</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
			<td style="width:108px;">Molecular Devices</td>
			<td style="width:119px;">Metamorph</td>
			<td></td>
		</tr>
		<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

単離された血液灌流肺製剤が広く可視化し、単一の微小血管内シグナリングを定義するために使用される。リアルタイムイメージングにより、この調製物を結合することによって、個々の肺微小血管透過性における変化を決定することが可能となる。ここに我々は、ラットの肺を隔離し、自己血でそれらを灌流する手順について説明します。その後、我々は小さな肺領域にマイクロカテーテルを経由してフルオロフォアまたは薬剤を注入するための手順の概要を説明します。記載され、これらの手順を使用して、我々は、細菌のリポ多糖の注入を受けてラット肺微小血管内の透過性の増加を決定した。データは、リポ多糖の両方細静脈や毛細血管の微小血管セグメントにわたって流体の漏れを増加させたことを明らかにした。したがって、この方法は、血管透過性のセグメント間の応答を比較することが可能となり、従って、応答内の任意の不均一性を規定する。肺の透過性を定義するために一般的に使用される方法は、肺組織サンプルの後処理を必要としながら、リアルタイムイメージングの使用は、本方法から明らかなように、この要件を回避する。このように、リアルタイムで画像化と組み合わせた孤立性肺準備が肺微小血管透過性を決定するために、従来の方法に比べていくつかの利点を提供しています、まだ開発し、実装するための簡単​​な方法です。

灌流チューブおよび関連機器に接続された単離された血液灌流肺製剤が図2に示されている。本明細書に記載された手順は、任意のネズミ種を用いることができるが説明のために、我々は、スプラーグドーリーラットを使用した。左心房マイクロカテーテルを通して注入は、肺のわずかな領域に到達する。注入された領域は注入によって誘発される変色（ 図3）によっ...

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

孤立した血液灌流肺準備が肺表面上の微小血管ネットワークを可視化することが実現可能になります。ここでは、リアルタイムの蛍光画像を用いて単離された肺における単一微小血管の透過性を定量化するためのアプローチを記載する。

単離された血液灌流ラットの肺の準備にシングル微小血管透過性を定量化する

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 ビュー.  The University of Tennessee Health Science Center. 孤立した血液灌流肺準備が肺表面上の微小血管ネットワークを可視化することが実現可能になります。ここでは、リアルタイムの蛍光画像を用いて単離された肺における単一微小血管の透過性を定量化するためのアプローチを記載する。 

ビデオ: 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 Dissociatorを使用してマウスの肺組織からの単細胞懸濁液の標準化の準備

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

生物学

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.

絶縁型、ベンチレーテッド、および計装のマウス肺の特性評価は、拍動流を灌流

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.

ザ ex vivoで</em血管反応性の調査のために>隔離された骨格微小血管の準備

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.

単離ミトコンドリアマウス心臓における透水性遷移孔の開口部のマルチパラメータ測定

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.

浸透水透過係数（Pの測定<sub&gt; F</sub&gt;）球状細胞の：例として単離された植物プロトプラスト

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.

新たに単離した糸球体の足細胞における単一チャネル分析とカルシウムイメージング

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.

ラット腸間膜に微小血管透過性の調節を調査するために、微小灌流法

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.

CD146の単離<sup&gt; +</supラット肺から&gt;常駐肺間葉系幹細胞

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.

マウス単離された灌流腎臓テクニック

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.

根の滲出液中の根茎細菌の化学誘引物質の迅速な同定のための改良された気化症アッセイ

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