The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.

倫理声明：マニトバ大学、ナイロビのケニヤッタ国立病院/大学の両方の研究倫理ボードは、この研究を承認し、書面によるインフォームドコンセントは、すべての研究参加者から得た。 1。メディアの作成およびCMCコレクションチューブ<ol><li>リン酸緩衝生理食塩水（PBS）溶液（137.93のNaCl、2.67 mMの塩化カリウム、8.1ミリモルのNa 2 HPO 4、1.47 mMのKH 2 PO 4）を調製する。無菌性のためのオートクレーブ。これは、数ヶ月間、4℃で保存することができる。 </li><li>事前に分量5ミリリットルのPBS 4ºCで50ミリリットルファルコンチューブ（サンプルあたり1を収集するため）、ストアにサンプル採取時まで。 </li><li>ストレージ用洗浄液を収集するために、サンプルあたり2-4凍結バイアルまたは1.5 mlマイクロチューブにラベルを付けます。 </li><li>細胞培養培地を調製する：RPMI 1640 + 1％ペニシリン/ストレプトマイシン/アンホテリシンB（終濃度100単位/ ml、100μg/ mlの250 ng / mlで、respectivイーリー、無FCSを/ FBSを追加しました。 </li></ol>細胞採取用ブラシのサンプルの2。コレクション細胞採取用ブラシサンプルの採取は、訓練されたMDや婦人科医によって行われなければならない非侵襲的な処置である。 <ol><li>細胞採取用ブラシ、木製やプラスチック製のスクレーパーの両方を使用して子宮頸核細胞（CMC）を収集します。 （ 図1）スクレーパーを挿入し、子宮口の周りに回転させることによって、膣鏡検査中の参加者から、CMCを収集します。 、子宮頸管OSに細胞採取用ブラシを挿入して360°回転し、すぐに5mlのPBSを含む50ミリリットルファルコンチューブに細胞採取用ブラシとスクレーパーの両方を置く。 </li><li>処理まで4℃で氷/上のサンプルを保管してください。採取後2時間以内に細胞生存率は、処理のサンプルを維持する。 </li><li>可能な場合は、末梢血単核細胞CMC分析のための比較器/制御として機能する（PBMC）を単離するためのグリーントップヘパリンバキュテナー内の一致した血液サンプルを採取。 </李&gt; </ol> CMCで3。分離二重の手袋で、無菌バイオセーフティキャビネット内で、バイオセーフティーレベル2実験室でサンプル処理を実行します。 <ol><li>収集中に、CMC試料としては、血液で汚染になることができます。最終的なサンプル中のPBMCの交絡混入を防止するために、調査から見える血液汚染を有するサンプルを除外します。により、CMC試料から単離したリンパ球の数が少ないと、マイナー血液汚染を簡単に、CMCリンパコンポーネントを圧倒することができます。 </li><li>ブラシから細胞を解離させるために45秒間細胞採取用ブラシとスクレーパーの両方を含む渦ファルコンチューブ。サンプルは、このプロセスの間に、泡状になることがあり;これは正常です。 </li><li>スクレーパーオフ任意の残りの材料をこすり、及び漂白液にスクレーパーを廃棄するように細胞採取用ブラシを使用しています。ブラシ/スクレーパーに付着したまま任意の細胞を収集するには、親指と人差し指の間にスクレーパーを圧迫する新鮮な手袋を使用しています。セルおよびPBSを同じ50mlチューブに収集することができるように、ブラシを下にスライドさせます。漂白剤溶液に細胞採取用ブラシを破棄し、手袋を破棄。 重要：各サンプルのために新しい手袋を使用してください。 </li><li>新しい50mlファルコンチューブに100μmのナイロン細胞ストレーナーを取り付けます。トランスファーピペットを用いて、サンプルチューブからPBS-細胞懸濁液を回収し、新鮮なチューブにストレーナーを通して濾過する。 </li><li>オリジナルのサンプルチューブに、RPMI 1640の5ミリリットルを追加します。トランスファーピペットを用いて、RPMIで試料管の側面を洗浄し、新しい収集管にフィルターを介して移す。だけでなく、RPMIでナイロンフィルターの底部を洗浄します。 </li><li>遠心分離機514 x gで10分間サンプル。これは、CMCペレットの脱落を防止するために、このステップの間に遠心ブレーキを切ったままにすることが重要である。 </li><li>優しくペレットを乱さないように注意しながら、チューブから上清を除去。ペレットサイズは​​非常に変化になります。その中で他の場合には、ペレットはほとんど見えかつ高度に透明である間、私試料ペレットを、上皮細胞の多数の存在に起因する非常に大きい。 </li><li>ファルコンチューブを穏やかに攪拌することによりペレットを再懸濁。サンプルを洗浄するために、PBSの5ミリリットルを追加します。 </li><li>無遠心ブレーキを514×gで10分間、再び遠心分離します。 </li><li>慎重に上清を廃棄し、上記のようにペレットを再懸濁。細胞（PBMCの凍結保存プロトコルを使用して）凍結保存、刺激または表現型フローサイトメトリーのどちらかの準備ができている。 </li></ol> 4。のCMC表面染色およびフローサイトメトリー<ol><li>ブロッキング溶液100μl中、ステップ3.10からペレットを再懸濁し、96ウェルプレートまたはFACSチューブに移すのいずれか。ブロッキング溶液（Fcγ受容体をブロックするには）次のようにレシピがある：1.8μlのマウスIgG（最終濃度0.2μgの/μL）、5μlのFBSおよび93.2μlのFACS洗浄（PBS +2％FBS）。染色は、ペレットの大きさが十分に小さい場合には、96ウェルプレートで行うことができるが、ペレットが日常的に大きい場合FACSチューブ中で行われる必要があり得る。それに応じて、抗体を滴定することが重要です。 </li><li> 4℃にて/氷上で10分間サンプルをブロック</li><li> FACSチューブに96ウェルプレート、または500μlのFACSウォッシュ（PBS +2％FBS）100μlで細胞を洗浄。低オンブレーキと4℃で10分間600×gで遠心分離（またはオフ、ローブレーキの設定が利用できない場合）。 </li><li>上清を除去し、攪拌により細胞ペレットを再懸濁。生存能力染色（ライブデッド固定可能な実行可能性染料）を追加します。製造業者の指示に従って生存色素を調製する。これは、各研究室/サイトメーターのセットアップで使用するために滴定する必要があるかもしれません。 4℃の暗所で30分間インキュベート</li><li> （96ウェルプレート/ FACSチューブ用）細胞をPBSで100μl/500μLを洗ってください。低/ブレーキなしで10分間600×gで遠心分離する。 supernatanを削除T再懸濁細胞。 </li><li>総容量100μlで、表面マーカー抗体のカクテルで細胞をインキュベートする。重要：以前のサンプル染色を各フローサイトメトリーパネルを最適化し、滴定する。 4℃の暗所で30分間、細胞をインキュベート</li><li>ステップ4.5を繰り返します。 FACSチューブに、96ウェルプレートに転送細胞を染色する場合、1％パラホルムアルデヒド（PFA）を含有するFACSウォッシュ750μlの最終体積に希釈する（またはのCytofixを、4分の1に希釈）。フローサイトメーターでのデータ収集に進みます。 </li></ol> 5。収集と子宮頸部膣肺胞洗浄液の調製（CVL） <ol><li>サンプリングを細胞採取用ブラシの前に、滅菌PBS 2mlで子宮頚内膜を洗浄し、円蓋から洗浄液を吸引するために注射器を使用しています。 </li><li> 15ミリリットルファルコンチューブに吸引洗浄サンプルを収集し、処理するまで4℃で氷/続ける。 </li><li>細胞破片を除去するために7分間400×gで遠心分離サンプル。 </li><li> CO上澄み、1ミリリットルまたは500μlのアリコートに分量のサンプルを、-70℃で保存llectアリコートCVLタンパク質のELISAまたはビーズアレイ解析のために解凍が、複数回の凍結/融解サイクルを回避することができる。 </li><li>所望であれば、製造業者のプロトコルに従って、RNA発現を測定するために後でRNA中の細胞破片のペレットを再懸濁。 </li></ol> 6。データ収集<ol><li>抗体パネルと一致する補償チューブのセットを準備します。スペクトルの重複を調整するために、補償管を実行します。そのサイトメーター任意の多色の流れにサンプルを実行するには、パネルで使用される蛍光色素標識抗体用に設定されている。 </li><li>リンパ球集団を検出するために、前方散乱（FSC）および側方散乱（SSC）の電圧を調整するように一PBMCサンプルを取得する。リニアスケールで、各軸の100でそれらを維持しようとします。 PBMC制御を実行すると、それは非常に簡単、CMCのリンパ球集団を見つけることになります。 </li><li> CMCサンプルのFSC閾値は低い値でFSC SSC軸をスミアによるPBMCサンプルに対して増加される必要があり得る。 </li><li>取得する前に、各チューブをボルテックスする。収集されたリンパ球ゲートイベントの数を最大化するために管全体を取得する。目詰まりを避けるために慎重にマシンを監視します。 </li><li>このようなFlowJoソフトとして、フローサイトメトリー解析プログラムにデータをエクスポートします。 </li></ol> 7。ゲー戦略<ol><li>一重の人口を決定するために、前方側方散乱高（FSC-H）/前方側方散乱面積（FSC-A）を選択します。 </li><li>買収の品質を制御するために、蛍光マーカー対時間（たとえば、FITCなど）を選択します。流量の変化は、データ内のアーティファクトを導入することができる。流量の不一致を示している任意の領域を除外します。 </li><li> SSC-A/FSC-Aプロット上のリンパ球集団を特定します。識別を容易にするために、PBMCコントロールを使用します。 CMCのリンパ球集団は、PBMCコントロールのように明確ではないかもしれないことに注意してください。&lt;/李&gt; <li> SSC-A/viability上のゲートは、生細胞から死細胞を排除するために染める。死んだ細胞は、非特異的に成果物を紹介しますこれは、抗体を組み込むことができます。 </li><li> T細胞集団を同定するSSC-A/CD3上にゲート。この門から、CD4 +およびCD8 +集団を特定します。 </li></ol>

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J., 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=HIV-exposed+seronegative+commercial+sex+workers+show+a+quiescent+phenotype+in+the+CD4++T+cell+compartment+and+reduced+expression+of+HIV-dependent+host+factors.">HIV-exposed seronegative commercial sex workers show a quiescent phenotype in the CD4+ T cell compartment and reduced expression of HIV-dependent host factors.</a> The Journal of infectious diseases. 202 Suppl 3, (2010).</li><li>Card, C. M., 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=Reduced+Cellular+Susceptibility+to+In+Vitro+HIV+Infection+Is+Associated+with+CD4T+Cell+Quiescence.">Reduced Cellular Susceptibility to In Vitro HIV Infection Is Associated with CD4T Cell Quiescence.</a> PloS one. 7, (2012).</li><li>Prodger, J. L., 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=Foreskin+T-cell+subsets+differ+substantially+from+blood+with+respect+to+HIV+co-receptor+expression,+inflammatory+profile,+and+memory+status.">Foreskin T-cell subsets differ substantially from blood with respect to HIV co-receptor expression, inflammatory profile, and memory status.</a> Mucosal immunology. 5, 121-128 (2012).</li><li>Reusch, L. M., 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=Nonlinear+optical+microscopy+and+ultrasound+imaging+of+human+cervical+structure.">Nonlinear optical microscopy and ultrasound imaging of human cervical structure.</a> Journal of biomedical optics. 18, (2013).</li><li>Oertelt-Prigione, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+and+the+menstrual+cycle.">Immunology and the menstrual cycle.</a> Autoimmunity reviews. 11, (2012).</li><li>Rahman, S., 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=Mucosal+serpin+A1+and+A3+levels+in+HIV+highly+exposed+sero-negative+women+are+affected+by+the+menstrual+cycle+and+hormonal+contraceptives+but+are+independent+of+epidemiological+confounders.">Mucosal serpin A1 and A3 levels in HIV highly exposed sero-negative women are affected by the menstrual cycle and hormonal contraceptives but are independent of epidemiological confounders.</a> American journal of reproductive immunology. 69, 64-72 (2013).</li></ol>

The authors have nothing to disclose.

女性の生殖器（FGT）の免疫に関する知識に大きなギャップを考えると、CMCで表現型分析は、子宮頸部の複数のリンパ球集団への洞察の広い配列を提供することができます。プロテオーム解析と子宮頸洗浄液中のウイルス量の測定と相まって、性感染症（STI）Sおよび他の病原体に対する免疫は、様々な集団で解剖することができます。 技術的な考慮-のCMC：CMC...

世界中の新たなHIV感染の大半は（UNAIDS 1）2011年に新たな感染の47％に相当女性と、異性間感染によって起こる。女性の生殖器（FGT）、HIVやその他の性感染性病原体のメインエントリポータルの1を理解すること、感染症を予防するための効率的な戦略を見つけることへのパスに非常に重要である。性器粘膜での免疫応答は、明らかに固有のものであり、末梢血2で測定されたものとは異なる。しかし、FGTの免疫動態の現在の知識は、最高の状態で制限されています。現在までに、粘膜免疫環境の研究は、主としてそれが感染後に粘膜組織における初期事象は、その後の疾患の進行3,4に強い影響を有することが明らかとなった腸管関連リンパ組織（GALT）に焦点を当ててきた。性器粘膜からサンプルを収集することは大きな課題であるとLACのために少なくとも部分的に責任があるFGTの免疫学の理解のK。 FGTはこのロケールからサンプルを収集し、分析するための効率的な方法が必要とされている特定の環境のコンテキストでのホストと病原体との間に免疫のダイナミックのパズルを解く。 FGTは2つのセクションに分かれています卵管、子宮内膜および子宮頚内膜が含まれ、上部生殖管および子宮膣部が含まれている下気道と膣（Kaushicによって審査ら 5）。これは、これらの異なる部位の相対的寄与はHIV感染症であるものはまだ不明であるが、両方の部位がHIVエントリー6に寄与し得ると考えられている。マクロファージ（ロドリゲス·ガルシアら 2に概説されている）約10％を構成しながら、T細胞は、上部および下部生殖管における白血球の40〜50％を表す。 T細胞は、膣、子宮頚部、および子宮内膜中で検出することができる。マクロファージはより強くlocaliですそれらが両方の組織で検出することができるが、子宮内膜および子宮頸部より子宮筋層の結合組織にゼット。最後に、形質細胞様樹状細胞（pDC）およびランゲルハンス細胞もFGT組織において検出することができる。表現型および免疫人口やHIV感染に対する感受性の割合がホルモンサイクルに応じて重要なのは異なる場合があり、ホルモン避妊薬、細菌性膣炎や性的活動5,7-9の使用。 多様な方法がFGTの免疫集団と環境を研究するために開発されてきた。子宮頸部生検、子宮頸cytobrushesと頸膣洗浄液（CVL）10月12日は、最も一般的に文献全体で使用されています。 PBSの洗浄によるCVLコレクションは、最も簡単な方法であり、極めて低い細胞収量の免疫調節タンパク質が、結果の研究を可能にするため、FGT 13の免疫細胞集団を研究するためには適していない。 CVLサンプルは、一方で、νある例えば、ELISA、サイトカインビーズアレイ14 15,16または質量分析などの方法を用いて種々のサイトカイン、ケモカインまたは抗菌因子の発現を測定することにより、FGTの免疫環境を評価するのに有用ERY。免疫細胞の頻度、表現型および機能の特徴付けは、子宮頸部細胞採取用ブラシによって、または子宮頸部生検サンプリングによって子宮頸部単核細胞（CMC）を収集することによって達成することができる。 子宮頸部生検標本は、出血の不快感や危険性を高めると女12の免疫状態に応じて、手順に従って癒すために2から11日かかる侵襲的な方法である。一方、子宮頸cytobrushesは、採取した細胞のより低い利回りにもかかわらず、FGTから免疫細胞を収集するための低侵襲、より便利な方法である。両方の方法は、CD45 +白血球の同じ収率に達することができるが、二つの連続子宮頸部cytobrushesが含まれる細胞と同じ量を得るために必要であるiをN 1の生検13。それにもかかわらず、細胞採取用ブラシのサンプリングは、まだ14フローサイトメトリーによるさらなるex vivoでの表現型解析のための細胞の許容数（約5000 CD45 +細胞/細胞採取用ブラシ）を提供します。刺激および細胞内フローサイトメトリーまたは定量PCRは、HIV特異的免疫応答17又はTh細胞の分極18を識別するために、細胞採取用ブラシ由来のCMCを使用して実施されているように、また、機能的な特徴付けは、これらの試料上で行うことができる。 T細胞集団の拡大はまた、CMCを19と機能的研究を容易にすることができる。 それは、生検およびcytobrushesはFGTの異なる部分をサンプリングしていることに注意することが重要である。子宮頸cytobrushesは子宮頸部、おそらく移行帯の上皮由来の細胞を集め、子宮口をサンプリングしながら、生検、子宮膣部12,13の上皮と間質の優れた部分に由来している。細胞採取用ブラシのサンプルは、そのための再サンプリング生検は、扁平重層上皮5によって裏打ち領域を含み、一方祇園は、円柱上皮の単層からなる。その結果、子宮頸部生検および細胞採取用ブラシによって収集白血球集団の性質が異なっている。 cytobrushesは、CD14 +単球/マクロファージ13の割合が高くのコレクションになるのに対し、生検では、CD3 + T細胞の割合が高く集める。 FGTの免疫学を研究することは、長年にわたって20〜22関心されており、我々は細胞採取用ブラシ由来のCMCの研究と専門知識の多くを蓄積してきました。我々の研究は、ケニアのナイロビからのHIVに感染し、感染していないと、HIV-曝露反応陰性（HESN）女性セックスワーカーの研究に主に焦点を当てています。 HIVは優先的にFGTでHIVの標的とすることができる活性化された細胞の23と下部の数字は、HIVの獲得に対する保護に貢献できる活性化したT細胞で複製。この仮説に沿って、いくつかのストゥディESは非常にHIVにさらされているHESNのセックスワーカーの免疫活性化は、まだ感染していない24,25まま下に記載されており、この静止表現型はまた、FGT 14で観察される。ここでは、処理のための方法論を記載し、ex vivoでフローサイトメトリーによって子宮頸部cytobrushes由来CMCサンプル中のT細胞の活性化を評価する。

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			<td height="21" style="height:21px;width:295px;">Name of Material/ Equipment</td>
			<td style="width:136px;">Company</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:205px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">100uM Cell Strainer for 50 ml Falcon tube</td>
			<td style="width:136px;">BD</td>
			<td style="width:133px;">352360</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">RPMI 1640</td>
			<td style="width:136px;">Hyclone</td>
			<td style="width:133px;">SH30027.01</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fetal Bovine serum&#160;</td>
			<td style="width:136px;">Life technology</td>
			<td style="width:133px;">16000044</td>
			<td>CMC processing</td>
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			<td height="21" style="height:21px;width:295px;">Fungizone</td>
			<td style="width:136px;">Life technology</td>
			<td style="width:133px;">15290-018</td>
			<td>CMC processing</td>
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			<td height="21" style="height:21px;width:295px;">Penicillin/streptomycin</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:133px;">P4333-20ml</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">50ml Falcon tube</td>
			<td style="width:136px;">Fisher</td>
			<td style="width:133px;">14-959-49A</td>
			<td>CMC processing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Blood Bank disposable transfer pipette</td>
			<td style="width:136px;">Fisher&#160;</td>
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			<td>CMC processing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cytobrush plus</td>
			<td style="width:136px;">Cooper surgical</td>
			<td style="width:133px;">C0121</td>
			<td>CMC sampling</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Disposable cervical scraper</td>
			<td style="width:136px;">Quick medical</td>
			<td style="width:133px;">2183</td>
			<td>CMC sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">15 ml Falcon tube&#160;</td>
			<td style="width:136px;">Fisher&#160;</td>
			<td style="width:133px;">14-959-70c</td>
			<td>CVL processsing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">1.5ml tube ependroff</td>
			<td style="width:136px;">Fisher</td>
			<td style="width:133px;">05-402-18</td>
			<td>CVL storage</td>
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		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Cell Stain Kit</td>
			<td style="width:136px;">Invitrogen</td>
			<td>Various</td>
			<td>Flow cytometry reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation Buffer (4% PFA)</td>
			<td style="width:136px;">BD</td>
			<td>554655</td>
			<td>Flow cytometry regeant&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">IgG mouse&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:133px;">I8765</td>
			<td>Flow cytometry regeant&#160;</td>
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collection, isolation, and flow cytometric analysis of human endocervical samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

マルチパラメータフローサイトメトリーは、以前に特徴づけられていない組織において、細胞サブセットの表現型や機能を分析するための強力なツールです。 CMC試料の分析は、適切なゲーティング戦略を、リンパ球および単球の両方の集団についての情報を得ることができる。 代表的なCMCゲーティング戦略は、一致したPBMCプロファイルと比較して、 図2に?...

Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

人間頸管サンプルの採取、単離、およびフローサイトメトリー分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

collection-isolation-flow-cytometric-analysis-human-endocervical

Research

JoVE Journal

Medicine

19.0K ビュー.  University of Manitoba. The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays. 

ビデオ: Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

部分的に膵臓を摘出された患者からのヒト膵島の単離

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

医学

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

ヒト膵臓の収集プロトコル

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

ヒト胎児膵臓細胞クラスターの単離、培養、およびイメージング

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

離と新鮮な外科的試料からのヒト心室の心筋細胞の機能解析

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

ヒト前立腺癌サンプルからがん幹細胞の単離

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

迅速な分別とコミュニティベースの設定から得られた試​​料中の全血液成分の単離

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

ヒト胆汁からマイクロRNA含有エキソソームの単離とプロファイリング

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

異なる均質化法を通じてマウス脳/脊髄から取り出された複数の細胞型の同時フロー細胞特性評価

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

ヒトの血流を模倣したフローループモデルにおける血液接触インプラントのヘモ適合性試験

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

小さな毛髪サンプルで11の薬剤耐性結核薬を定量化するための検証済みのLC-MS/MSパネル

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...