著者は、技術支援のためにミスリアナMackiewiczに感謝したいと思います。この作品は、ビクトリア州政府業務インフラのサポートとオーストラリア政府は国立保健医療研究評議会IRIISSとプロジェクト無償1031212により可能となった。

1。 P.によるマウスの感染ベルゲイ -ANKA <ol><li>霜凍結保存Pのアリコートベルゲイ ANKA PRBC。 </li><li>両手拘束技術を用いて、脳マラリア耐性のBALB / cドナーマウス（8-12週齢）を抑制する。 1mlのインスリン注射器（28G針）を使用して、PRBCの100から200μlでマウスを注入します。日常的に、1-2ドナーマウスが注入される。 </li><li>感染後4-5日で（pi）をケージからドナーマウスを削除して、ワークステーションまたは使い捨て作業パッドの上に置きます。 </li><li>ゆっくり尾の端でマウスを抑制し、小さなハサミを使用する（約1mm）尾の先端をカットします。代わりに、25 Gの針を使って小さな尾穿刺を行うことができた。 </li><li>曇らされたエンド顕微鏡スライドの終わり近くにマウスの尾静脈から血液を一滴。 </li><li> 45°の角度で第2のスライド（スプレッダー）を配置し、一滴の血にそれをバックアップしてください。血液はエッジに沿って広がりたら、スプレッダー-スライド前夜をプッシュ血液塗抹標本を作るために顕微鏡スライドを越えnly。スミアが空気乾燥させてください。 </li><li> 30秒間100％メタノールに血液塗抹標本を修正してから10分間、新たに調製した染色でスライドを染色する。 </li><li> 1分間流水で血液塗抹標本をすすぎ、空気乾燥することができますし、顕微鏡（100倍、油浸）の下のスライドを調べる。千赤血球内PRBCを列挙し、パーセント寄生虫を計算します。彼らは実験動物の感染症のために血を流したことができる前にドナーマウスは百分の2.5から5の間に寄生虫のレベルに到達する必要があります。 </li><li>ヘパリン処理マイクロヘマトクリット毛細管を用いて眼窩出血によってドナーマウスから血液を採取します。全ての実験は、ウォルター·エリザホール研究所動物倫理委員会の要件に準拠して行われます。地元の動物倫理委員会の要件に応じて、他の出血の手順は、使用されるかもしれません。 1X10 6 pRBC/0.2 mlの最終濃度でRPMI培地中で血液の必要量を溶かす。それを考えてみましょう正常なマウスのヘマトクリット値は、〜6×10 9赤血球/ 1ミリリットルです。 </li><li>腹腔内（ip）に1×10 6 pRBC/0.2ミリリットルを注入することにより、実験のC57BL / 6マウス（8-12週齢）に感染。 </li><li>感染マウスの寄生虫レベルを監視するために、ステップ1.3から1.8に従ってください。寄生虫は、2日目または3πから始まる2〜3日ごとに決定されるべきである</li><li> C57BL / 6マウスにおける重症マラリアの発症は猫背外観、エリマキ毛皮や低活性として現れることがあります。一部のマウスには、この段階で回復するかもしれませんが、自己立ち直り反射の消失への進行が不可逆的な疾患や動物を安楽死させなければならないことを示します。 </li></ol> 2。マウスと脳抽出の心臓内灌流<ol><li> 60 cmのカラムスタンドの上部に取り付けられたカラムホルダーに500mlの貯水池を確保することにより、手動重力灌流システムを組み立てる。貯水池の底端部にプラスチックのチューブを接続し、バッファの流れを制御するためのチューブクランプを固定します。 23Gの針にチューブを取り付けます。 FILPBSでリザーバー（22℃に保た℃）までのlは、クランプとバッファはすべての気泡を除去するためにチューブを介して実行できるように開きます。 </li><li>安楽死P. CO 2吸入によってベルゲイ ANKA感染マウス。ペダル、軌道や呼吸器の応答の有無によって死亡を確認します。 </li><li>背プラスチックトレイに格納されている発泡スチロールの解剖ボード上の後ろ足と前足で安楽死させたマウスをピン。地元の動物倫理委員会の要求に応じて麻酔が心臓内pefusionの事前の開始を投与することができる。 </li><li> 70％エタノールで腹側を拭きます。 </li><li>胸腔を露出するように正中線に沿って皮膚を開くために、大きなハサミとピンセットを使用しています。両側に折り、ピン肌。 </li><li>細かい鉗子で胸骨を押しながら、横隔膜をカットし、胸骨の両側に沿ってリブを切断する。任意の大血管を傷つけないように注意してください。 </li><li>頭の横に緩く胸骨によって胸郭をピン。 </li><li>心室のウィットを保持hは細かい鉗子と細かいはさみで慎重に切開右心房。 </li><li> PBSが実行されている間上行大動脈に向かって左心室に重力灌流システムに接続されている23Gの針を挿入します。針先端のわずか0.5センチメートルを挿入します。 </li><li> 5分間または排水が透明になるまでマウスを灌流する。 </li><li>固定を解除し、マウスとその腹部の上に横たわっていた。 70％エタノールで頭を拭いてください。 </li><li>大きなハサミを使って、ちょうど頸髄領域の上のカットを行う。 </li><li>頭蓋骨を露出するために離れて正中仙骨-吻側頭蓋底から始まるカット、剥離皮膚を作るために微細なハサミを使用します。 </li><li>頭を抱え、それぞれの軌道の空洞に小さなハサミの刃を配置し、軌道の間のカットを行います。 </li><li>矢状縫合に沿って長手方向の切り込みを入れ、慎重に外側にそれぞれの脳半球上の頭蓋骨をはがします。 </li><li>へらを使って、そっと脳を持ち上げ、RPMI培地を含む10mlの遠心管に入れてください。 </li></ol>3。脳隔離白血球の単離（BSL） <ol><li>安全キャビネットでの作業、場所がたてたRPMI組織培養培地の3-5 mlを入れたペトリ皿の中でのステンレス鋼のセルストレーナー（40-60メッシュサイズ）の上に脳を収穫した。 </li><li>小片に脳組織を切り取ります。 </li><li>クリスタルプランジャーを使用してセルストレイナーで脳組織の小片を押してください。 </li><li> 4℃で10分、250×gで10 mlのチューブと遠心分離機に脳ホモジネートを転送</li><li> 0.05パーセントコラゲナーゼDと2U/ml DNアーゼIを含むRPMI培地3ml中でペレットを溶解</li><li>室温で30分間混合物を回転させます。 70μmナイロンセルストレイナーでおよび/または5分間氷上でインキュベートすることにより、混合物を押すことにより、細胞の破片を取り除きます。 </li><li>室温で20分間、400×gで（ノーブレーキ）で7ミリリットル30％パーコールクッションと遠心分離機に脳ホモジネートを置きます。 </li><li> 5分〜氷上で赤血球溶解バッファーとインキュベート1ml中のペレットを再懸濁し心臓内灌流により脳から削除できませんでした付着PRBCを、溶解する。 </li><li> RPMI培地を9mlを加え、4℃で10分間、250×gで遠心分離し、細胞を洗う</li><li> RPMI培地とエッペンドルフチュー​​ブに移す細胞の50〜100μLに再懸濁し、BSL-含有ペレット。 </li><li>生細胞の同定のためにトリパンブルーで細胞サンプルを1:2の5〜10μlのアリコートを希釈します。 </li><li>血球計数器を用いて顕微鏡下で生細胞をカウントします。 6日目以降はπ、Pからベルゲイ ANKA感染マウスは20,000-100,000 BSLsを回収することができる、神経症状が表示されます。 </li></ol> 4。マルチカラーフローサイトメトリーによるBSLの免疫表現<ol><li> 4℃で10分間、250×gで遠心し、染色緩衝液50μl（PBS、1％ウシ胎児血清（FCS）、2mMのEDTA）に懸濁し、上清とペレットを吸引除去する。 </li><li> anti-CD16/32抗体（Fcblock）を精製し、1μlを添加する。 </li><li> 1細胞をインキュベート氷の上で0分。 </li><li>染色緩衝液1mlで細胞を洗浄します。 4℃で10分間、250×gで遠心し、上清を吸引除去する。 </li><li>必要な蛍光抗体、 すなわち抗CD4、抗CD8、抗TCRと抗NK1.1の所定の最適な希釈を含む染色緩衝液50μlの再懸濁します。 </li><li>氷上で1時間インキュベートする。 </li><li>染色緩衝液1mlで細胞を洗浄します。 4℃で10分間、250×gで遠心し、上清を吸引除去する。 </li><li> 100μlのPBSに細胞を再懸濁する。 </li><li>フローサイトメトリー用プラスチックチューブに細胞を移します。 </li><li>フローサイトメーターを使用して、少なくとも5,000〜10,000のイベントを取得する。必要に応じて適切な染色細胞のコントロールおよび蛍光補正のサンプルが含まれるべきである。 </li><li>適切なフローサイトメトリー·ソフトウェアを使用して結果を分析します。 </li></ol>

<ol>
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特別な利害関係は宣言されません。

BSLの単離と解析、特性評価および実験マウスモデルにおける組織損傷または感染に反応して脳への移行の炎症性細胞の定量化を可能にする方法です。臓器の抽出とそれに続く細胞単離の前に血液区画の除去のための心臓内灌流ステップの導入は、非炎症性の循環白血球と炎症細胞の汚染を防止するのに有用である。これは、炎症細胞が脳実質5を貫通しているようなマウス脳?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td></td><td></td><td></td><td> ソリューションおよびバッファ </td></tr><tr><td>ギムザアズールエオシンメチレンブルー液</td><td>メルクミリポア</td><td> 1.09204.0500 </td><td>蒸留水で1:10に希釈</td></tr><tr><td> RPMI培地</td><td></td><td></td><td>マウス張</td></tr><tr><td>マウス張PBS </td><td></td><td></td><td> 20 mMリン酸ナトリウム、0.149のNaCl、pH7.3の</td></tr><tr><td> 0.4％トリパンブルー</td><td>シグマアルドリッチ</td><td> T-8154 </td><td> 1:2希釈</td></tr><tr><td>コラゲナーゼD </td><td>生化学ワージントン</td><td></td><td></td></tr><tr><td> Deoxyribonuclease（DNアーゼ）私</td><td>シグマアルドリッチ</td><td> D4263-5VL </td><td>ウシ膵臓由来</td></tr><tr><td>パーコール</td><td> GEヘルスケア</td><td> 17-0891-01 </td><td> PBS中30％溶液</td></tr><tr><td>純トリス</td><td>インビトロジェン</td><td> 15505-020 </td><td></td></tr><tr><td>塩化アンモニウム（NH 4 Cl） </td><td> AnalaR </td><td> 10017 </td><td></td></tr><tr><td>赤細胞溶解バッファー</td><td></td><td></td><td> 17 mMトリス、14nMのNH 4 Cl、pH7.2の</td></tr><tr><td> FCS </td><td>ギブコ</td><td> 1009 </td><td></td></tr><tr><td> EDTA二ナトリウム塩</td><td>メルク</td><td> 10093.5V </td><td> 0.1M、pH7.2の</td></tr><tr><td></td><td></td><td></td><td> 抗体とコンジュゲート </td></tr><tr><td>抗マウスCD16/CD32（FCブロック）、クローン2.4G2 </td><td> BDファーミンジェン</td> &lt;TD&gt; 553142 </td><td> 50μLの染色緩衝液で1μL（0.5mg/50ミリリットル） </td></tr><tr><td> FITC-抗マウスCD4、クローンH129.19 </td><td> BDファーミンジェン</td><td> 553651 </td><td></td></tr><tr><td> PE-抗マウスNK1.1、クローンPK136 </td><td> BDファーミンジェン</td><td> 553165 </td><td></td></tr><tr><td> PerCPCy5.5抗マウスCD8、クローン53-6-7 </td><td> BDファーミンジェン</td><td> 551162 </td><td></td></tr><tr><td> APC-抗マウスTCR-β、クローンH57-597 </td><td> BDファーミンジェン</td><td> 553174 </td><td></td></tr><tr><td> PE-抗マウスCXCR3、クローン220803 </td><td> R＆Dシステムズ</td><td> FAB1685P </td><td></td></tr><tr><td>ビオチン化抗マウスCCR5、クローンC34-3448 </td><td> BDファーミンジェン</td><td> 559922 </td><td></td></tr><tr><td>ストレプトアビジン-も、PerCP-Cy5.5 </td><td> BDファーミンジェン</td><td> 551419 </td><td></td></tr><tr><td></td><td></td><td></td><td> 機器と材料 </td></tr><tr><td> SuperFrost顕微鏡スライド</td><td>ボシュロムメンツェル-グレイザー</td><td></td><td></td></tr><tr><td>解剖用ピンセット、はさみ</td><td> REDA Instrumente </td><td></td><td></td></tr><tr><td> 500ミリリットルのPBS貯水池</td><td>ナルゲン</td><td></td><td></td></tr><tr><td>ゴムチューブ</td><td></td><td></td><td></td></tr><tr><td> 23G針</td><td> BD PrecisionGlide </td><td> 302008 </td><td></td></tr><tr><td>金属ふるいを含む細胞解離キット</td><td>シグマアルドリッチ</td><td> CD-1 </td><td></td></tr><tr><td> 70μmのナイロン細胞ストレーナー</td><td> BDファルコン</td><td> 352350 </td><td></td></tr><tr><td>血球計</td><td> GmbHはノイバウアー</td><td> 717810 </td><td></td></tr><tr><td>フローサイトメトリー管</td><td> BDファルコン</td><td> 352008 </td><td></td></tr></tbody></table>

isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice

我々はPの脳血管から付着炎症細胞の単離および特性評価のための方法を説明ベルゲイ ANKA感染マウス。実験的脳マラリア、人間1,2における熱帯熱マラリア原虫媒介重症マラリアの再現する一定の重要な側面、その神経学的症候群の誘導にこの寄生虫のひずみの結果と感受性マウス系統の感染。血液段階のマラリアの成熟型は、それらが血管内皮細胞に結合することができます感染赤血球の表面に寄生するタンパク質を発現している。このプロセスでは、低酸素症および出血3、その結果、血流に障害を誘発し、また寄生虫隔離のサイトに炎症性白血球の動員を刺激する。 他の感染症とは異なり、すなわちneutrotopicウイルス4-6、両方マラリア寄生赤血球（PRBC）ならびに関連inflammatory白血球は血管内ではなく、脳実質に浸潤するより隔離のままである。したがって、非炎症性の循環細胞、臓器の抽出と組織の処理の前に感染したマウスの大規模な心臓内灌流による隔離白血球の汚染を避けるために、血液区画を削除する場合は、この手順が必要になります。灌流後、脳を採取し、小片に解剖した。組織構造は、さらにその結果脳ホモジネートは、その後ミエリンおよび他の組織片から脳隔離された白血球の分離（BSL）が可能パーコール勾配に遠心分離されるコラゲナーゼDとDNase Iで酵素処理によって破壊されています。単離された細胞は、その後、フローサイトメトリーによってその後の分析のための蛍光抗体と血球計数器とステンドを使用してカウントし、洗浄する。 この手順では、炎症性白血球の包括的な表現型の特徴は、再の脳への移行を可能にするストロークだけでなく、ウイルスや寄生虫感染症を含む様々な刺激に応答特性。この方法はまた、前臨床動物モデルにおける新規抗炎症治療の評価のための便利なツールを提供します。

図の結果。 2に示す比率と灌流したりunperfusedマラリアに感染し、ナイーブ対照マウスの脳から回収された別のBSLの人口の絶対数。議定書のテキストに示されているように隔離されたBSLは、PE-抗NK1.1およびAPC-抗TCR-β抗体で染色した。 7月9日以前の知見と一致して、αβTCR+ T細胞は、灌流しマラリア感染マウス（6日目PI）の脳におけるBSLプールの高い割合を占めている。この集団?...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

の脳血管から付着炎症性白血球を単離するための方法<em&gt;マラリアベルゲイ</em&gt; ANKA感染マウスが記載されている。この方法は、フローサイトメトリーによって蛍光抗体とその後の分析で染色した後に定量化ならびに単離された白血球の表現型の特徴を可能にします。

脳から隔離された白血球の単離と解析<em&gt;マラリアベルゲイ</em&gt; ANKA感染マウス

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

isolation-analysis-brain-sequestered-leukocytes-from-plasmodium

Research

JoVE Journal

Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

12.7K ビュー.  The Walter and Eliza Hall Institute of Medical Research. の脳血管から付着炎症性白血球を単離するための方法マラリアベルゲイ ANKA感染マウスが記載されている。この方法は、フローサイトメトリーによって蛍光抗体とその後の分析で染色した後に定量化ならびに単離された白血球の表現型の特徴を可能にします。

ビデオ: Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

脳浸潤白血球の分離

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

免疫・感染学

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

自然送信時に結核マラリア重複感染を研究する実験モデル結核菌とマラリアベルゲイ</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

ヒト白血球と血小板の単離と評価のためのプロトコル：生体エネルギーと酸化バースト

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

フローから白血球の補充に間質細胞の影響を分析する

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

ヒト母体·胎児のインターフェイスから白血球の単離

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

母体·胎児界面におけるマウス組織からの白血球の分離

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

酵素消化および勾配分離を使用したマウス皮膚から浸潤白血球の単離

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

弱毒で皮内免疫後のマウスの皮膚及び排出リンパノードから骨髄性細胞の単離<em&gt;マラリア原虫</em&gt;スポロゾイト

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating肺白血球の選択的ハーベスティング

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-肝白血球の選択的ハーベスティング

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...