我々は、マウス操作のヘルプについては超遠心分離し、パティカイザーのヘルプはスティーブMcBryantとジェフハンセンに感謝。国立衛生研究所の神経疾患と脳卒中の国立研究所、助成金5R01NS056379 - 02は、この作業に資金を供給した。

<html> 1。プリオンロッドを精製し、ラベル付けこのプロトコルは、1つの以前に発行された18から適応される<ol><li>コマーシャルブレンダーの最大速度で1分、その後、氷上で2分間氷冷均質化緩衝液900mLの中のプリオン感染脳組織の100グラム（HB、1X PBS、320 mMスクロース、150mMのNaClおよび4mm EDTAを含む）をホモジナイズする。 3回繰り返します。 </li><li> 10 3000 × gで分間、4℃でホモジネート遠心する氷の上で削除し、上清を保存する。 1L HBでペレットを再サスペンドし、手順1.1と1.2を繰り返します。 </li><li> 10万XG、0℃で60分間、プールの上清と遠心上清を捨てる。 </li><li>再サスペンド1Xトリス100ml中のペレットは、2％トリトンX - 100を（TBST）を含む（10mMトリス- HCl、0.1 mM NaClおよび1mMのEDTA）緩衝生理食塩水。ブラッドフォードまたは類似のアッセイによる推定のタンパク質濃度とTBSTで5 mg / mLのために調整する。 30分間氷上で冷やします。 </li><li> 10万XG、0℃で30分間遠心上清を廃棄し、50 mLのTBSTで100mLのTBSのペレットを洗浄する。 100 mLの1X PBSでペレットが1％サルコシルとプロテアーゼ阻害剤カクテルを含む再サスペンドし、37℃で120分間攪拌℃に</li><li> 10万xgで60分間、320 mMスクロースと遠心900mLの上の層のサンプルを、10℃上清を捨て、10mLの2.3MのNaCl、5％サルコシルで再サスペンドペレット。 </li><li>氷上で1分間の間隔で70％の最大パワーで試料5 × 40秒を超音波処理してください。 </li><li> 13000 × gで、4℃で15分間エッペンドルフチュー​​ブと遠心分離機への試料のアリコートを1mL。 TBSと店で二回ペレットを洗浄@ -70 ° Cまたはステップ1.9に蛍光色素にコンジュゲート。 </li><li> DyLight 649の1バイアルを使用して、プリオンロッドを精製の共役1チューブは、製造業者のプロトコールに従って蛍光色素。 </li><li>フィルタ列を介して透析や遠心分離によるPBSのための交換DyLightの標識バッファー。 -70℃で離れて使用する準備が整うまで、光から20μL分注しで保管してください。 </li></ol> 2。プリオン接種と細胞回復このセクションでは、腹腔内プリオン接種と腹膜、縦隔および腸間膜リンパ節および脾臓からの細胞の回復を包含する。 <ol><li>使用直前に蛍光プリオンロッドPBSで1:50に希釈する。親指と人​​差し指で背首の毛皮でクラフのマウスは、優しくあなたを直面し、小指でテールを抑制するためにマウスを回す。頭の上に後部胴体を昇降し、わずかに上下逆に彼を保持し、マウスを回外させる。 </li><li> 30Gのインシュリンの注射器を使用して、仙腸関節前方正中線および1-2 cmの1センチメートル横蛍光プリオンロッド100μLを注入する。皮膚を通して針1 cmの内側に45 °を指示挿入。コントロールとして蛍光ビーズまたはPBSのみの1:50希釈を使用してください。分析の前に割り当てられた時間のためのマウスをインキュベートする。 </li><li>承認された動物の世話に応じてマウスを安楽死させると委員会のプロトコルを使用してください。 12mLのシリンジと25G針を用いて腹腔内にPBS 10 mLを注入する。ゆっくりと1分間のために縦方向に死体を揺らし。死体の解剖を継続しながら氷の上に15 mLコニカルチューブと予備に同じシリンジと16G針を用いて腹膜から細胞懸濁液を回復する。 </li><li> 70％エタノールで十分に死体の腹側を濡らす。ちょうどピンセットで胸郭下の正中線で皮膚を持ち上げて、はさみで皮膚を通して小さな切開をカット。そこからは、それぞれの端から横方向に正中線から2台の2センチ切開し、両方向でから頭と尾の皮膚を介して3〜5センチメートル長い切開を行います。バックピールと腹膜と胸部の空洞を明らかにするために肌をピン。慎重に腹膜を取り巻く薄い半透明の膜を通ってカットし、縦隔を明らかにするために胸部を反映している。 </li><li>気管の腹側に近い腕頭動脈の内側に隣接してわずかに背側と胸腺への横方向に隣接する2から4縦隔リンパ節を、、、見つけて削除します。検索と後腹壁にアンカー腸をその柔らかな白い腸間膜の組織に埋め込まsix腸間膜リンパ節、最大取り外します。脾臓、ちょうど正中線の外側と腹部の左側の腸への背側に位置する、長い、暗い赤色の臓器を見つけて削除。氷上で1 mLのRPMI 1640培地で予備のリンパ節郭清が完了するまで。 </li><li>温浸法と1 mLシリンジのプランジャーを使用してプラスチック製のペトリ皿にRPMI 3mlに40μmのメッシュのストレーナを介してリンパ節を押してください。 RPMIの追加2mLでストレーナーをすすぎ、15 mLコニカルチューブに5 mLの細胞懸濁液を移す。 RPMIのさらに5 mLでペトリ皿をすすぎ、同じチューブに移す。 250 × gで5分間遠心分離、上清を、1mLのFACSバッファー（1X PBS、1％ウシ血清アルブミン及び10mMのEDTA）に細胞ペレットを再サスペンドし、1.5mlのエッペンドルフチュー​​ブに移す捨てる。 </li></ol><html> ove_title&quot;&gt; 3。細胞染色およびフローサイトメトリー以下は、よく特徴免疫細胞表面マーカーに対する蛍光抗体を用いた免疫細胞のサブセットを識別するための典型的な染色のプロトコルです。すべての抗体は使用直前にFACS緩衝液に希釈した。 <ol><li>エッペンドルフチューブに細胞を、アリコート10 6個の細胞を数える。遠心細胞（2.7参照）と1mLのFACS緩衝液で洗浄ペレット2X。 </li><li>内因性のFc受容体をブロックするには、2 ng / mLのラット抗マウスCD16/32の100μLで細胞を氷上で20分間インキュベート。ペレットとFACS緩衝液で細胞を洗浄する。 </li><li>氷上で1時間ごとにサンプルとインキュベートする1​​ng / mLの適当な蛍光不格好なやつ（Y）IES 100μLを加える。細胞を制御するために単独でFACS緩衝液100μLを加える。ペレットとFACS緩衝液で細胞を2回洗浄する。 </li><li>細胞を1 mLのACKバッファー（150mMのNH 4 Clを 、1mMのKHCO 3、0.1mMのEDTA）赤血球を溶解するために1分間氷上でインキュベートで再懸濁する。ペレットとFACS緩衝液で細胞を洗浄する。 1 mLのFACS緩衝液で細胞を再懸濁する。 </li><li>通常、405、488および633nmの励起レーザーと五から十蛍光発光検出器で、多色検出や分析が可能なフローサイトメーターを使用してセルからデータを収集する。 </li></ol> 4。代表的な結果： 我々は、原油、プリオン感染脳乳剤（ 図1）また、プロテアーゼKだったE2ヘラジカ脳ホモジネート（ レーン1および2）大きく（ レーン1と3を比較する ）プリオンの棒の濃縮、の界面活性剤可溶化および超遠心分離からプリオンロッドを精製抵抗性（レーン4）。興味深いことに、未消化の精製プリオンロッドは、プリオンのための劇的な濃縮を示す、消化E2の脳ホモジネートと非常に類似グリコフォームプロファイルを表示。同じ処理された正常な脳ホモジェネートはPK耐性PrP Cを （ レーン5および6）産生しなかった。 特定の細胞集団のフローサイトメトリー分析は、その抗原は二時間で腹膜に細胞のホームを提示し、蛍光ビーズとプリオンロッド、などのPBSを注入コントロール上に劇的に増大した蛍光（ 図2）によって証明さを保持示す。重要な蛍光を表示ビーズから細胞（ 図2H - N）とプリオン（ 図2O - U）接種マウス中にバックグラウンド（ 図2A - G）の上に蛍光表示を制御するPBS処置の腹膜から採取さない細胞なし、、特に単球上に（ 図2IおよびP）、樹状細胞（ 図2KおよびR）とマクロファージ（ 図2LとS）、およびいくつかの好中球（ 図2JとQ）と少ないB細胞（ 図の2T）。ビーズとプリオン有利子単球、樹状細胞やマクロファージは、2つの時間接種（ 図2ADとAK、AFおよびAMとAGと 、それぞれ）、一方、ほとんどまたは全くのPrP CWD有利子好中球の後縦隔リンパ節に認められた、BまたはT細胞は、（それぞれ、 図2AEとAL、AHとAGとAI及びAP、）そこに発見された。我々は、脾臓や腸間膜リンパ節（データは示していない）にはビーズやプリオンのロッドを検出されませんでした。トトでは、これらのデータは、他の粒子状抗原と非常によく似たその免疫細胞のトラフィックのプリオンを示しています。 <table border="1"><tbody><tr><td> 試薬 </td><td> 蛍光色素 </td><td> 励起レーザー（波長） </td><td> ピーク発光（nm）の </td></tr><tr><td>プリオンロッド</td><td> DyLight 649 </td><td> 633 </td><td> 674 </td></tr><tr><td> 1μmのビーズ</td><td> AlexaFluor 660 </td><td> 633 </td><td> 685 </td></tr><tr><td> 抗体 </td><td></td><td></td><td></td></tr><tr><td> CD11b </td><td> eFluor 450 </td><td> 405 </td><td> 450 </td></tr><tr><td></td><td>フィコエリトリン- Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> CD11cは</td><td> R -フィコエリスリン</td><td> 488 </td><td> 575 </td></tr><tr><td></td><td>フィコエリトリン- Cy7 </td><td> 488 </td><td> 760 </td></tr><tr><td> Ly6C </td><td> Fluoroisothiocyante </td><td> 488 </td><td> 518 </td></tr><tr><td> Ly6G </td><td> R -フィコエリスリン</td><td> 488 </td><td> 575 </td></tr><tr><td> CD21 </td><td> DyLight 488 </td><td> 488 </td><td> 518 </td></tr><tr><td> B220 </td><td>アロフィコシアニン- Cy7 </td><td> 633 </td><td> 785 </td></tr><tr><td> CD3 </td><td> R -フィコエリスリン</td><td> 488 </td><td> 575 </td></tr></tbody></table>蛍光試薬の &quot;&gt; 表1。スペクトル特性 <img src="/files/ftp_upload/2349/2349fig1.jpg" alt="図1" /> 図1。感染した脳ホモジネートからプリオンロッドの精製は、我 々は我々が集約されたプリオンロッド（レーン3および4）、精製、そこから出発材料としてCWDに感染したシカ（E2、レーン1と2）から10％の原油の脳ホモジネートを使用。正常な脳のホモジェネートていないのに対し（レーン5および6）、 - 原油と精製材料の両方は、プロテアーゼK処理（4レーン1）に特徴的な抵抗性を示した。分子量マーカーは、ブロットの左側にKdがで表示されます。 BH、脳のホモジネート。 <img src="/files/ftp_upload/2349/2349fig2.jpg" alt="図2" /> 図2。二時間接種。PBS（パネルAGとV - AB）、蛍光ビーズ（HNおよびAC - AI）またはプリオンロッドの後腹膜からの縦隔リンパ節への免疫細胞の人身売買のプリオンのフローサイトメトリー分析 （OUとAJ - AP）があった二時間後に腹腔洗浄液（AU）や縦隔リンパ節（V - AP）から収穫マウスと細胞の腹膜内に注入。最初の列のグラフは、PBS（パネルとV）、蛍光ビーズ（パネルHおよびAC）とプリオンロッド（パネルOとAJ）で処理したマウスからの細胞を示す。蛍光細胞（赤または緑の点）と総細胞（グレードット）の相対的な大きさ（前方散乱）、粒度（側方散乱光）と蛍光を発する総生細胞の割合を示すためにプロットされます。顆粒球の特徴的な細胞の重要な数字は、蛍光ビーズやロッドを保持。細胞はまた、免疫細胞表面マーカーとLYG - Ly6C +単球のためのゲート（パネルB、I、P、W、ADとAK）、Ly6c - CD11cは、CD11b + Ly6G +好中球（C、J、Q、X、AEに対する抗体で染色したとAL）、Ly6G - Ly6C - CD11b + CD11cは+樹状細胞（D、K、R、Y、AFおよびAM）、Ly6G - Ly6C - CD11cは、CD11b +マクロファージ、（E、L、S、Z、AGと）、CD3 - B220 + CD21 + B細胞（F、M、T、AA、AHおよびAO）とCD21 - B220 - CD3 + T細胞（G、N、U、AB、AIおよびAP）。単球、樹状細胞やマクロファージは腹腔（パネルIとP、KとRとLとS、それぞれ）で蛍光ビーズとプリオンロッドを保持し、縦隔リンパ節（パネルADとAK、AFおよびAMとAGにそれらを輸送そして、それぞれ）、同様の取り込みとこれら二つの粒子の人身売買を実証。好中球は、ビーズのかなりの量（J）と少ないロッド（Q）を保持されますが、リンパ節（AEおよびAL）に配信するために失敗しました。 B細胞は、いくつかの棒（TとAO）が事実上ないビーズを（MおよびAH）が保持して輸送。 T細胞はビーズもロッド（N、U、AIおよびAP）のいずれも人身売買。

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-derived+dendritic+cells+acquire+and+degrade+the+scrapie+agent+following+in+vitro+exposure.">Skin-derived dendritic cells acquire and degrade the scrapie agent following in vitro exposure.</a> Immunology. 116, 122-122 (2005).</li><li>Gilch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CpG+and+LPS+can+interfere+negatively+with+prion+clearance+in+macrophage+and+microglial+cells.">CpG and LPS can interfere negatively with prion clearance in macrophage and microglial cells.</a> FEBS J. 274, 5834-5834 (2007).</li><li>Safar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mass,+biochemical+composition,+and+physicochemical+behavior+of+the+infectious+form+of+the+scrapie+precursor+protein+monomer.">Molecular mass, biochemical composition, and physicochemical behavior of the infectious form of the scrapie precursor protein monomer.</a> Proc.Natl.Acad.Sci.U.S.A. 87, 6373-6373 (1990).</li><li>B&#252;eler, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+devoid+of+PrP+are+resistant+to+scrapie.">Mice devoid of PrP are resistant to scrapie.</a> Cell. 73, 1339-1339 (1993).</li></ol>

利害の衝突は宣言されません。

ここでは、大幅に周辺プリオンの感染の初期段階でイベントを監視して容易にin vivoでのタグ付けと追跡プリオンのためのプロトコルを示す。このプロトコルは非常に明確に内因性のPrP Cからそれを区別するために事前にラベルの高濃縮プリオンの接種による in vitro 9 および in vivo 3,12 におけるモニタリングのプリオンの摂取で過去の試みを?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CWD-infected elk brain</td>
<td>&nbsp;</td>
<td>Private elk farm in Colorado</td>
<td>&nbsp;</td>
<td>Use any non-human prion-infected brain</td>
</tr>
<tr>
<td>Blender</td>
<td>&nbsp;</td>
<td>Oster</td>
<td>6694-015</td>
<td>Use any commercial blender</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>SS34 rotor</td>
<td>Use any centrifuge /rotor that can reach 3000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>50.2 Ti rotor</td>
<td>Use any ultracentrifuge /rotor that can reach 100,000 x g and hold &ge; 500 ml volumes</td>
</tr>
<tr>
<td>Bradford Reagent</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B6916</td>
<td>&nbsp;</td>
<tr>
<td>Complete mini protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>&nbsp;</td>
<tr>
<td>Sonicator</td>
<td>&nbsp;</td>
<td>Misonix</td>
<td>MP4000X</td>
<td>Use any horn or probe sonicator set to ~70% max power</td>
</tr>
<tr>
<td>DyLight antibody Labeling kit</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>53050</td>
<td>&nbsp;</td>
<tr>
<td>microcentrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>55430R</td>
<td>Use any refrigerated microcentrifuge that can achieve 13,000x g</td>
</tr>
<tr>
<td>centrifugal filter columns</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>Microcon YM-100</td>
<td>Use any filter or dialysis membrane with 100 Kd molecular weight cutoff</td>
</tr>
<tr>
<td>8-40 week-old FVB mice</td>
<td>&nbsp;</td>
<td>Charles River</td>
<td>207</td>
<td>Use any inbred mouse strain</td>
</tr>
<tr>
<td>1 &mu;m red fluorescent beads</td>
<td>&nbsp;</td>
<td>Phosphorex</td>
<td>2307</td>
<td>Use any fluorescent bead &le; 10 &mu;m</td>
</tr>
<tr>
<td>RPMI 1640 medium </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875-093</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>fluorescent antibodies</td>
<td>&nbsp;</td>
<td>BD pharmingen</td>
<td>Various</td>
<td>Use any fluorescent antibody appropriate for your application.</td>
</tr>
<tr>
<td>flow cytometer</td>
<td>&nbsp;</td>
<td>Dakocytomation</td>
<td>CyanADP</td>
<td>Use any flow cytometer capable of multicolor fluorescence detection</td>
</tr>		</tbody>
		</table>
		</div>

monitoring immune cells trafficking fluorescent prion rods hours after intraperitoneal infection

異常な形態が存在するプロテアーゼ、抵抗性病態と感染しているホストでエンコードされたプリオン蛋白質（PRPC）は、羊のcervidsとスクレイピーの慢性消耗病（CWD）などのプリオン病を特徴付ける。プリオン仮説は、この異常なコンフォーマーは、ほとんどまたはすべての感染性プリオンのを構成すると主張する。周辺プリオン病因の早期イベントの免疫システムの役割は、説得力CWDとスクレイピー1-3実証されている。マウスのトランスジェニックおよび薬理学的研究は、感染4月6日後の早期にプリオンを保持し、複製における補体系の重要な役割を明らかにした。in vitroおよび in vivo研究における人身売買の彼らの役割は残っても、7-10樹状細胞によるプリオンの保持を観察している11月16日は不明。マクロファージは、同様に初期のプリオンの病因に関与している、しかしこれらの研究は、感染3,11,17後数週間に発生した事象に焦点を当てている。これらの先行研究はまた、内因性プリオンCと感染プリオン区別の問題に苦しんでいる。ここでは、そこに採用免疫細胞で接種部位からプリオンの取り込みと人身売買を評価するための半定量的、公平なアプローチを説明します。集約されたプリオンロッドは、非集約タンパク質と糖クッションを介して超遠心分離の界面活性剤可溶化により、感染した脳ホモジネートから精製した。ポリアクリルアミドゲル電気泳動、クマシーブルー染色とペレット画分に高濃縮プリオンロッドのウェスタンブロッティング確認回復。プリオンロッドは、蛍光色素で標識した後、マウスに腹腔内に注射した。二時間後に腹腔洗浄液、脾臓および縦隔および腸間膜リンパ節から免疫細胞は、プリオンロッドの保持および単球、好中球、樹状細胞、マクロファージやB細胞とT細胞のマーカーを使用して多色フローサイトメトリーで識別される細胞のサブセットを測定した。このアッセイは、感染後数時間以内に生体内で取得し、人身売買のプリオン免疫細胞の初めての直接モニタリングすることができます。このアッセイはまた、明らかに、通常は困難であると他のアッセイシステムでデータの解釈の問題が発生する可能性が宿主細胞に発現PRPCから感染、集約されたプリオンを区別します。このプロトコルは、他の接種経路（経口、静脈内、intranervousや皮下など）と同様に抗原（結合ビーズ、細菌、ウイルスおよび寄生虫病原体やタンパク質、卵）に適合させることができます。

Watch this Scientific Journal Video about Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection at JoVE.com

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

ここでは、すぐに精製することにより腹腔内接種に従い、蛍光して注射部位からの彼らの取り込みと動きを監視し、これらのイベントを媒介する細胞を特徴づける感染脳材料から集約されたプリオンロッドを標識する免疫細胞によるプリオンの取り込みと人身売買を監視するための新たなアッセイを説明します。

腹腔内感染後に免疫細胞の人身売買蛍光プリオンロッドの時間を監視

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

monitoring-immune-cells-trafficking-fluorescent-prion-rods-hours

Research

JoVE Journal

Immunology and Infection

11.4K ビュー.  Colorado State University. ここでは、すぐに精製することにより腹腔内接種に従い、蛍光して注射部位からの彼らの取り込みと動きを監視し、これらのイベントを媒介する細胞を特徴づける感染脳材料から集約されたプリオンロッドを標識する免疫細胞によるプリオンの取り込みと人身売買を監視するための新たなアッセイを説明します。

ビデオ: Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

フローサイトメトリーで固定し、生細胞におけるカルパイン活性の測定

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

免疫・感染学

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

マウス大動脈内の免疫細胞のフローサイトメトリー分析

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

機能心臓の免疫細胞の単離

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

機能と分子生物学的研究のためのプライマリマウスII型肺胞上皮細胞のフローサイトメトリ分離

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪組織の免疫細胞の単離

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

フローサイトメトリーによる一次免疫細胞亜集団との蛍光ナノ粒子の相互作用を検出

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

ヒト末梢血の免疫表現型分析のための抗体のカクテルを準備する半自動のアプローチ

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...