我々は、技術支援のためのバージニアドハーティとJunmei八尾に感謝します。この研究は、KAGにNIMH（R01 MH084966）、および米国陸軍研究室と米国防総省の国防高等研究計画庁（グラントW911NF-10-1-0059）によって資金を供給されました。

全ての実験は、成体雄ロングエバンスラットを用いて行きました。すべての手順は、米国国立衛生研究所に応じて、実験動物の管理と使用に関する（NIH）ガイドであり、マサチューセッツ工科大学の施設内動物管理使用委員会との動物管理使用レビュー庁によって承認されましたUSAMRMC。 1.準備<ol><li>ヘパリン（千USP単位/ ml）を含む500μlのチューブでシールド針を配置した後、針を介してヘパリン溶液を吸引して排出することによりHepariniseカテーテルとシリンジ。 <ol><li>シリンジに蝶カテーテルを取り付けます。損傷から鋭い先端を保護するために、カテーテルの針の上にシールドしてください。 </li><li>収集される血液の量よりもわずかに大きいヘパリンの量を撤回。注射器を外し、空気でそれを埋めます。 </li><li>再アタッチカテーテルに注射器を使用し、空気は、過剰なヘパリン溶液を排出します。確実に微量のみ、チューブ、針、シリンジに残ります。 </li><li>滅菌表面に、ついたまま注射器で、滅菌カテーテルを配置します。 </li></ol></li><li>すぐに前足と後足が快適な位置にあり、呼吸が制限されていないことを保証きれいな布でラットを固定します。 <ol><li>フックとループテープでラップを固定し、外性器がくびれていないことを確認してください。 </li><li>優しくアシスタントを持ち、しっかりと尾はカウンターの端にぶら下がっで堅実な仕事の表面上にラット（腹部と尾の付け根）を抑制する。 </li></ol></li></ol> 2.採血<ol><li> 40〜50秒の血管を拡張し、ペーパータオルで尾を乾燥させるために42℃の水に尾を浸し。 （尾をねじる防止するために、尾と体全体を回転させる）から採血する尾静脈の位置を確認します。 注：尾の十分な温暖化が急速collectioのために重要です血液サンプルのN。血管系の狭窄された場合、カテーテルの適切な配置が困難であり、血流が大幅に低減されます。加熱パッドは、水浸漬の代わりに使用することができます。 </li><li>サンプリング点を特定します。 注：動脈は尾の半ば背側面に沿って位置します。サンプリングのためにこれを使用しないでください。 <ol><li>動脈への側方に位置する左右の尾静脈のいずれかをターゲットにしています。菌株によって異なり、年齢とともに増加する尾の色素沈着は、血管系の一部を覆い隠すことができます。尾の下部に静脈の一部を対象としています。 </li><li> 2％クロルヘキシジン消毒液でターゲット領域を拭いてください。 </li></ol></li><li>約50μlにゼロからプランジャーを引き出すことによって、注射器やカテーテル内の負圧を作成します。 </li><li>ストレートサンプル収集を通じて尾を維持するために先端付近優しく、しっかりと尾を持ちます。血流が過度にタイトなグリップによって閉塞されていないことを確認してください。</li><li>ゆっくりと約5cm、尾の先端から浅い角度で静脈にカテーテルを挿入します。静脈が貫通されると、血液がカテーテル内に流れます。ゆっくりと一定の速度（毎秒〜20μL）で所望の体積を収集するために、注射器のプランジャーを撤回。 <ol><li>収集することができる最大の血液量については、獣医のスタッフにご相談ください。収集する必要があります血液の最大量は、ラットの体重と健康状態に依存します。 14日の期間中の総血液量の15％以上を撤回しないでください。 注：血液は急性ストレスホルモンが血管系を収縮するので、コレクションをサンプリングする前に数分で強調された動物から収集するためにはるかに困難です。例えば、動物をラップするのに数分を取って、新規の部屋にラットのホームケージを移動、または静脈にカテーテルの挿入を繰り返し、すべての急性ストレス反応を引き起こす可能性があります。 </li><li> blooを促進「搾乳」静脈からdフロー。尾の先端に向かってベースから、静脈の長さに沿って指を実行しますが、挿入された針やカテーテルの先端から2cm以上が静脈から除去になる可能性が残っています。 </li><li>血液が正常にカテーテルの浸透の最初のサイトから収集することができない場合は、静脈までさらに針を再挿入します。血液は最初のサイトで収集された場合は、切断した後、静脈に先立って再挿入するカテーテルと注射器を再接続することにより、針を再加圧します。一般的には、追加の貫通を避けます。 </li><li>複数の貫通は、尾への血液供給が遮断され、柔らかい尾組織を壊死されるようになる中で、尾静脈の崩壊を引き起こす可能性がありますように尾静脈の崩壊がある場合に、ラットを安楽死させます。 </li></ol></li><li>十分な試料体積が収集されると、切断したカテーテルを再接続することによって、注射器内の圧力を解放します。吸引液がわずかにシリンジプランジャを使用して（〜50＆＃181; L）、および静脈から針を撤回。 注意：針が第1のシリンジ内の圧力を解放することなく撤回された場合、血液が針から滴下します。 </li><li>簡単に説明すると出血を止めるために挿入部位に圧力を適用し、消毒液で拭きます。そのホームケージにラットを返します。 </li></ol> 3.血液サンプルを処理<ol><li>吸引した空気には血液がカテーテル針の内部に残っていないことを確認し、ちょうど針の上にカテーテルチューブをカットするはさみを使用しています。滅菌1.5ミリリットルのマイクロチューブに血液を放出します。 注：血液が針を通して押された場合、剪断力は、多くの下流のアッセイを妨害し得る破裂する赤血球を引き起こす可能性があります。溶血を回避するために、針を外します。 <ol><li>血漿、ここで（抗凝固剤としてEDTAが含まれている血液の200〜400μlの0.1 M EDTA10μlのを使用し、使用チューブを収集するには、使用されるEDTAの濃度は、Wに干渉しないよう下流アッセイ番目）、氷上に置きます。 <ol><li>回収の10分以内に10分間冷却遠心機（4℃）で2,100×gでの全血サンプルをスピン。赤および白血球層を乱す避け、プラズマを溶出します。 </li></ol></li><li>凝固を可能にするために最大30分間、室温で（抗凝固剤を含まない）は、血液、血清、場所のサンプルを収集します。 2,000×gで冷却遠心機（4℃）で回収チューブをスピン。血清は、その後、溶出され得ます。 </li></ol></li><li>使用直ちに試料、または1年まで-80℃で保存。 </li></ol>

<ol>
	<li>Vausort, M., Wagner, D. R., Devaux, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+Noncoding+RNAs+in+Patients+with+Acute+Myocardial+Infarction.">Long Noncoding RNAs in Patients with Acute Myocardial Infarction.</a> Circ Res. 115 (7), 668-677 (2014).</li><li>Shah, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+for+Early+Detection+of+Colorectal+Cancer+and+Polyps:+Systematic+Review.">Biomarkers for Early Detection of Colorectal Cancer and Polyps: Systematic Review.</a> Cancer Epidemiol Biomarkers Prev. 23 (9), 1712-1728 (2014).</li><li>Chan, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+blood-based+protein+biomarker+strategies+in+the+study+of+psychiatric+disorders.">Applications of blood-based protein biomarker strategies in the study of psychiatric disorders.</a> Prog Neurobiol. , (2014).</li><li>Cao, L., Li, L., Lin, D., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induces+learning+impairment+that+is+mediated+by+interleukin+1beta+in+rodents.">Isoflurane induces learning impairment that is mediated by interleukin 1beta in rodents.</a> PLoS One. 7 (12), e51431 (2012).</li><li>Culley, D. J., Baxter, M. G., Yukhananov, R., Crosby, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+impairment+of+acquisition+of+a+spatial+memory+task+following+isoflurane-nitrous+oxide+anesthesia+in+rats.">Long-term impairment of acquisition of a spatial memory task following isoflurane-nitrous oxide anesthesia in rats.</a> Anesthesiology. 100 (2), 309-314 (2004).</li><li>Vahl, T. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+ACTH+and+corticosterone+sampling+methods+in+rats.">Comparative analysis of ACTH and corticosterone sampling methods in rats.</a> Am J Physiol Endocrinol Metab. 289 (5), E823-E828 (2005).</li><li>Kalinichenko, L. S., Koplik, E. V., Pertsov, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+of+peripheral+blood+in+rats+with+various+behavioral+characteristics+during+acute+emotional+stress.">Cytokine profile of peripheral blood in rats with various behavioral characteristics during acute emotional stress.</a> Bull Exp Biol Med. 156 (4), 441-444 (2014).</li><li>McEwen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+effects+of+stress+hormones+in+health+and+disease:+Understanding+the+protective+and+damaging+effects+of+stress+and+stress+mediators.">Central effects of stress hormones in health and disease: Understanding the protective and damaging effects of stress and stress mediators.</a> Eur J Pharmacol. 583 (2-3), 174-185 (2008).</li><li>Sanchez, A., Toledo-Pinto, E. A., Menezes, M. L., Pereira, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+norepinephrine+and+epinephrine+concentrations+in+adrenal+gland+of+the+rats+submitted+to+acute+immobilization+stress.">Changes in norepinephrine and epinephrine concentrations in adrenal gland of the rats submitted to acute immobilization stress.</a> Pharmacol Res. 48 (6), 607-613 (2003).</li><li>Meyer, R. M., Burgos-Robles, A., Liu, E., Correia, S. S., Goosens, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ghrelin-growth+hormone+axis+drives+stress-induced+vulnerability+to+enhanced+fear.">A ghrelin-growth hormone axis drives stress-induced vulnerability to enhanced fear.</a> Mol Psychiatry. , (2013).</li><li>Knoll, A. T., Carlezon, W. 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著者らは、開示することは何もありません。

ここでは、他の一般的に使用される技術上の重要な利点がありますラットから血液サンプルを得るための迅速かつ簡単な手順について説明します。まず、頚静脈または眼窩後洞からのサンプリングとは対照的に、麻酔を必要としません。血液サンプルは、行動手順を取り巻く収集された場合には、学習および記憶4,5と干渉することができるので、麻酔薬の投与は望ましくありません。第二に、そのような伏在や背ペダル静脈から収集などの他の静脈穿刺技術、より大きな血液容量を収集する機能を提供しています。血液1.5 mlまでの、ここに記載の技術を使用して、単一の時点でラットから回収することができる、容易に複数のアッセイを並行して実行されることを可能にする量。最後に、この手順では、尾の先端切断または眼窩後出血と比較して、組織の損傷の可能性を最小限に抑えることができます。この手順の使用は、動物Wの遵守を促進します動物で実施される実験方法から生じる痛みや苦痛を最小限に抑える必要が法および実験動物の管理と使用に関する指針は 、elfare。 それは、この方法に新しい研究者は、実験動物が抑制されている時間を最小限に抑えるために拘束し、尾の出血技術を練習することをお勧めします。拘束の開始から3分未満で採取した血液は、最適な結果を提供します。 ここで説明するプロトコルは、週に1〜4回のサンプリングに使用されないが、一日二回よりも多くすることができます。繰り返し血液収集を行うことができるが、尾の基部から上方に移動する別のサンプリング部位を使用する必要があり、左右の尾静脈サンプリング部位として交互にされるべきです。げっ歯類の総血液量は、その体重の6〜7％であり、全血液量の15％以下は、2週間の期間内に収集されるべきではありません。血清または血漿を採取したサンプルの体積の約40〜60％を含みます。 いくつかの小さな変更を加えてここで説明したように外側尾静脈を介して血液採取は、マウスで実行されてもよいです。まず、わずかなゲージ（27 G）カテーテルを使用することができます。第二に、それは、マウスを固定化するために、むしろラップよりも、チューブ拘束具を使用することをお勧めします。顎下維管束（200〜500μL）の静脈穿刺を用いて、マウスから得ることができる血液の量を安全に尾静脈（200μlの最大値）から採取することができるよりも大きいです。顎下維管束からのサンプリング血液が最小限の拘束を必要とし、より多くの血液をもたらす可能性があるため、これは、マウスでのサンプリングのための好ましい経路です。 この手順は、その低侵襲性の性質と共に、実行することができるこれで迅速に、また、急性ストレス反応6によって血液ベース措置の潜在的な摂動を最小限に抑えることができます。ザ急性ストレス反応は、インターロイキンおよび他の免疫活性因子7を含む多くの分子の循環レベル、視床下部-下垂体-副腎軸8のホルモン、交感神経系9、グレリン10、内因性オピオイド11、ドーパミンでホルモンを変えることができ、セロトニン12。これらの分子によって調節されるこれらの分子または他の静止循環対策が望まれる場合には、ストレッサ露光開始の分だけ少なく内でトリガされるストレス応答を最小限にすることが重要です。 ストレス反応は、血液の組成を変化させるだけでなく、交感神経系から増加したドライブを介しているため、血管系の収縮の血液採取のための技術的な障害を表すだけではなく。これは、急性のストレス応答をマウントされたラットからの安定した血流量を得ることが困難になる大きくします。したがって、動物の苦痛はミニムでなければなりません急速に関心のある生理的状態を反映し、サンプルを得るために化さ。

血液から得られたバイオマーカーは有用な診断、予測を提供し、心血管疾患1、がん科学2、および精神疾患3を含む多くの臨床状況において対策を、階層化。また、飢餓、炎症、またはストレスの存在度を含む、生物の「状態」を評価するために基礎科学において使用され得ます。このような措置は、またはサンプルが得られる時刻と対象者の性別など、関心の問題に重要であってもなくてもよいの変数によって影響を受けることができます。また、血液採取手順自体の間に誘起される応力によって影響され得ます。ストレスホルモンと痛みの知覚は急速に血液の組成を変化させることができます。 げっ歯類は、最も一般的に使用される実験動物であり、そして複数の方法は、血液採取のために開発されています。採血の理想的な方法は、最小限のphysiologicaを持つ必要があります動物のLへの影響は、全く麻酔を必要とせず、迅速かつ繰り返しサンプリングを可能にし、多数の下流の適用のために十分な血液量を提供します。例えば、頸静脈または尾先端切断のカテーテルなどの血液を採取するための一般的な技術は、これらの基準を満たしていません。 このプロトコルの目的は、最小限のストレスであるラットにおける使用のための血液サンプリング技術について記述して麻酔を必要とせず、単一の被験者内で複数の血液採取を可能にし、複数のアッセイを行うことができるように、比較的大きな試料体積を提供します単一のサンプルに。この方法の目的は、最低限、急性ストレス反応に影響される血液サンプルを得ることです。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium heparin (1000 USP units/ml)</td>
			<td>Patternson Veterinary Supply</td>
			<td>25021040010</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>JT Taylor</td>
			<td>JT2020-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dermachlor Rinse-Chlorhexadine</td>
			<td>Butler Schein</td>
			<td>6356</td>
			<td>Topical antiseptic solution, 2% chlorhexidine gluconate</td>
		</tr>
		<tr>
			<td>SURFLO Winged Infusion Sets, Terumo, butterfly catheters</td>
			<td>VWR Scientific</td>
			<td>TESV25BLK</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Tuberculin 1cc syringes</td>
			<td>VWR Scientific</td>
			<td>BD309659</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>VWR Scientific</td>
			<td>89202-682</td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#956;l microcentrifuge tubes</td>
			<td>VWR Scientific</td>
			<td>21150-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, stainless steel, 5&#34;</td>
			<td>VWR Scientific</td>
			<td>82027-586</td>
			<td></td>
		</tr>
		<tr>
			<td>500ml plastic beaker</td>
			<td>VWR Scientific</td>
			<td>414004-149</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean cloth wrap</td>
			<td>Butler Schein</td>
			<td>2993</td>
			<td></td>
		</tr>
		<tr>
			<td>Velcro tape, .75&#34; width</td>
			<td>Monoprice</td>
			<td>B004AF9II6</td>
			<td>Hook and loop tape</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>VWR Scientific</td>
			<td>62344-641</td>
			<td></td>
		</tr>
	</tbody>
</table>

sampling blood from the lateral tail vein of the rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

プロトコールに記載されているように外側尾静脈から採取した血漿は、外観に半透明で淡黄色であった血漿サンプルを提供します。 図1に示すように、サンプル中の溶血は、血漿に赤い色合いを与えます。急性ストレス反応が急速に血液の組成を変化させることができます。 図2に示すように、例えば、循環コルチコステロン濃度が著しく、ストレッサー曝露の10分以内に増やすことができます。露出をストレッサーの前に、この方法で得られたコルチコステロンの低い基礎レベルは、サンプリング手順自体がストレスの大きな原因ではないことを明らかにしました。 <img alt="図1" src="/files/ftp_upload/52766/52766fig1.jpg" /> 図1：サンプルの外観（A）A溶血試料が示されています。遠心分離後、血漿または血清の層（表面黒Arで示されます行）ピンクや赤を帯び表示されます。暗い色合いは、溶血の高いレベルを示しています。 （B）遠心分離した後、適切に収集したサンプルは、非溶血血漿または血清に対応する上のバンド（黒矢印で示す面）に透明な、黄色がかった外観を持つことになります。この層を除去するとき、それは、いずれかの全血層にピペットの先端を押して、またはチップ内に全血を吸引することにより、下層の全血を乱さないことが重要です。全血で汚染された任意の血漿または血清は破棄されるべきです。 <img alt="図2" src="/files/ftp_upload/52766/52766fig2.jpg" /> 図2：血漿コルチコステロンは急速にストレスの多い経験以下に上昇する血液が前に成人女性ロングエバンスラットの外側尾静脈から得られ、10分4トーン（10秒、2 kHzの、85デシベル）への曝露後の共同終了しました。 fooでtshocks（1秒、350μA）。ベースライン（290.4±138.8 pg / mlで）における血漿コルチコステロンのレベルは、フットショックストレスの提示後10分で観察されるよりも有意に低かった（2204.8±454.5 pg / mlで、p = 0.02、n = 4） は 、対応のあるtによって決定されます-test。 *、p &lt;0.05

Watch this Scientific Journal Video about Sampling Blood from the Lateral Tail Vein of the Rat at JoVE.com

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

ラットの外側尾静脈から血液をサンプリング

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

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Research

JoVE Journal

Biology

101.8K ビュー.  Massachusetts Institute of Technology. Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

ビデオ: Sampling Blood from the Lateral Tail Vein of the Rat

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

伏在静脈を通って心臓穿刺によりラットから採血

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

生物学

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

RNAまたは特定の脳領域からのタンパク質抽出用のラット脳の微小解剖

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

StemCell Technologies社からRosetteSepキットと人間の血液からNK細胞の濃縮

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

アメリカカブトガニ、カブトガニポリフェムスから採血

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

生体力学とMechanobiological研究ラット尾腱の準備

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

新生児マウスからの肺血管内皮細胞の単離と培養

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

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 ...

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

モルモットにおける非ターミナル採血テクニック

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

エピソームおよびHDAC阻害剤を用いて血液からの効率的なiPS細胞の生成

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for detecting the successful establishment of blood chimerism in parabionts by a caudal vein injection of glucose. Parabiotic mice were constructed. Glucose was injected into the donor mouse through the tail vein, and the fluctuation of blood glucose level was measured in both mice using a blood glucometer at different time points. Our results showed that after glucose injection, the blood glucose level in donor mice increased sharply after 1 min and decreased slowly thereafter. Meanwhile, the blood glucose level of the recipient mice peaked 15 min after injection. Similar results were obtained with Evans blue, used as a positive control for glucose. The synchronous fluctuation of blood glucose levels indicates that blood flow between the two mice was established successfully.

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

コーダル静脈グルコース注射によるパラバイオティクスマウスにおける共有血液供給の検出

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for ...