この作品は、衛生研究所（NIH）の国立研究所によってサポートされていましたR01-DK080752およびP50-DK064540を付与します。私たちは、このアッセイを確立する彼女の努力のためにJ·ロフマンに感謝します。

1。 5637膀胱上皮細胞を培養層流の組織培養フードUV照射を用いて調製し、70％エタノールで拭いで次の手順を実行します。 <ol><li>水浴中で10％ウシ胎児血清（FBS）を含むRPMI-1640培地を調製[RPMI +称される]、フィルターは、0.22μmの孔径のフィルターを用いて滅菌し、37℃に加温し。 </li><li> 37℃の水浴中で、5637細胞（膀胱癌由来アメリカン·タイプ·カルチャー·コレクションHTB-9）のクライオバイアルを解凍する。すぐに20ミリリットルのRPMI +を含む75cm 2の組織培養フラスコに解凍した細胞を移す。 </li><li>細胞が約95％コンフルエント（フラスコあたり約6×10 6細胞）を、約4日になるまで、5％CO 2の加湿雰囲気中で37℃でフラスコをインキュベートする。 </li><li> 5637細胞を継代培養するには、次のように<ol><li>フラスコから培地を除去します。 10ミリリットルダルベッコリン酸緩衝サリンで細胞を洗浄室温でのE（DPBS）。 </li><li> 6ミリリットル温かい（37℃）0.05％トリプシンをフラスコに0.02％EDTA溶液を添加し、15分間、5％CO 2、37℃でインキュベートする。 </li><li> 15ミリリットルコニカルチューブに細胞を移し、5分間300×gでの遠心分離によってペレット化。トリプシン/ EDTA溶液を除去します。 </li><li> 6ミリリットルRPMI +（10 6細胞/ ml）で細胞を再懸濁し、20mlのRPMI +を含む新しい75cm 2のフラスコに転送1ミリリットル（10 6細胞）。 </li></ol></li><li> 4日ごとに、または細胞が95％コンフルエントに達する細胞を継代培養を繰り返して手順1.4。 </li></ol> 2。透過性支持体上に5637細胞を播種し、成長無菌法を用いて組織培養フードで次の手順を実行します。最適な結果を得るために、10未満のサブカルチャーを受けた5637細胞を使用しています。 <ol><li>滅菌ピンセットを使用して、無菌の25ミリメートルの深さの組織培養皿に透過性のサポートを反転。 </li><l私は&gt; 1.4.1-1.4.3の手順に従って、95％コンフルエンスで5637細胞の75cm 2のフラスコをトリプシン処理。 </li><li> 1,000μピペットを用いて、穏やかに血球計数器を用いて細胞計数によって決定3×10 6細胞/ mlの濃度まで〜2ミリリットル+ RPMI中で細胞を再懸濁する。 </li><li>膜に接触することなく、各透過性支持に細胞懸濁液50μl（1.5×10 5細胞）を適用します。皿に蓋を置き、慎重にこれ以上の16以上の時間のために、5％のCO 2、37℃での皿を置く。 </li><li>ウェルあたり0.6ミリリットルのRPMI +を含む24ウェルプレートに滅菌ピンセット、右透過性担体を用いる。各透過性支持の上部タンクに0.1ミリリットルのRPMI +を追加します。 5％のCO 2、37℃でインキュベートする。 </li><li> 2日ごとに培地を交換してください。最初、吸引下池に続く上部貯水池からの培地、および、低い貯水池（0.6ミリリットル）に、逆の順序で新鮮な、RPMI +を適用してから、上RESErvoir（0.1ミリリットル）。 </li><li>七日5637細胞と浸透支援を播種後、細胞の合流点を評価。 、RPMI +（〜0.35ミリリットル）を上部貯水池を埋める。媒体は、上下のリザーバとの間に平衡しない場合に細胞がコンフルエントに十分である。 </li></ol> 3。細菌接種の準備<ol><li>無菌法を用いて、キャップを250ミリリットルのフラスコに、適切な培養液20ミリリットルを追加。 E.のためのルリア-ベルターニ（LB）培地を使用してください大腸菌の文化、そして適切な場合には培養液に抗生物質を追加します。 </li><li>無菌接種ループを使用して、グリセロールストックからの細菌やストリークプレートと培養液に接種。 UPEC株について、（1型線毛の産生を促進するために）、振とうすることなく、約16時間、37℃で細菌培養物をインキュベートする。 </li><li>遠心管に細菌培養を転送します。 10分間8000×gでの遠心分離によって細菌をペレット化。 </li><li>上清をデカント、および〜10 9 CFU / mlのと同等の、600 = 1.000にOD PBSに細菌を懸濁します。 <ol><li>熱殺菌細菌刺激を生成するには、30分間55℃で再懸濁した細菌の一定分量（150μL中の例えば 〜1.5×10 8 CFU）をインキュベートする。細菌の死を確認するために、熱殺菌懸濁液の一部をメッキ。 </li></ol></li><li>使用直前に再懸濁させ、細菌を（ライブまたは熱殺菌）10倍暖かい無血清RPMI中8 CFU / mlの10には、マイクロチューブ内に[RPMI-呼ば]希釈する。 </li></ol> 4。末梢血からヒト好中球の分離成人ボランティアからの採血は、事前審査や治験審査委員会の承認を必要とします。適切な個人保護具を着用し、適切にヒトの血液への暴露をさけるため、有害物質を処分。 <ol><li>健康な成人vから静脈血を約25ミリリットルolunteerは瀉血の訓練を受けたスタッフが3無菌ヘパリンナトリウム含有採血管に引き込まれるべきである。 <ol><li>しっかりと肘の上、上腕にゴム止血帯を包む。 </li><li>無菌70％アルコール拭きを使用して、侵入部位、前肘窩を消毒。 </li><li>翼付き翼状針システムにプラスチック製のチューブホルダーを取り付けます。 </li><li>上を向いて傾斜、30°の角度以下で肘正中静脈に針を挿入します。血液のスパートは、プラスチックチューブで表示されたときに針が静脈に穿刺しています。 </li><li>プラスチックチューブホルダ内に採血管を挿入する。最初のコレクションチューブが満杯になると、第2の収集チューブに置き換える。第3のチューブを繰り返します。 </li><li>第三のコレクションチューブは約半分いっぱいになったときに、止血帯を削除します。 </li><li>無菌コットンボールで穿刺部位をカバーし、徐々に静脈から針を引き抜く。針と場所、私の上に保護シールドをスライドさせバイオハザードシャープコンテナNA。 </li><li>穿刺部位に圧力を加える。絆創膏で穿刺部位や綿球をカバーしています。 </li><li>穏やかにヘパリンを分散させるために採血管を反転させる。 </li></ol></li></ol>無菌法を用いて組織培養フードで次の手順を実行します。 <ol start="2"><li> 10ミリリットルピペットを用いて、優しくフレッシュな滅菌50ミリリットルコニカルチューブに血液を移す。 3％に相当する容量を追加します（w / v）の0.9％NaCl中のデキストランおよび転倒混和。 20分間室温で直立管をインキュベートする。 </li><li>下層を中断させることなく、慎重に上層を吸引し、新しい50ミリリットルコニカルチューブに移す。 10分間300×gでの遠心分離により細胞をペレット化。上清を捨てる。 </li><li>血液の出発体積に相当する0.9％のNaClの容量で細胞ペレットを再懸濁する。 </li><li>細胞懸濁液に、p下で密度遠心分離溶液10mlレイヤ2つの相の間の界面を予約する。ブレーキなしで30分間400×gで遠心分離する。上清を捨てる。 </li><li>残りの赤血球を溶解するために10mlの冷0.2％NaCl中で細胞ペレットを再懸濁する。 30秒間インキュベートした後、速やかに等張性を復元するために10ミリリットルの冷1.6％のNaClを追加します。 </li><li> 6分間300×gでの遠心分離により細胞をペレット化。上清を捨てる。 </li><li>細胞ペレットは、赤血球、溶解の典型的には3回がないことが表示されるまで繰り返して、4.6から4.7を繰り返します。 </li><li> 1,000μピペットを用いて、温かい（37℃）RPMI-に血球計を用いて細胞計数によって決定10 7細胞/ mlの濃度で、主としてPMN、細胞ペレットを再懸濁。使用するまで37℃で細胞を維持する。それぞれ、染色後の核の形態を、トリパンブルー排除および可視化により評価されるように、典型的には、PMN生存率および純度は&gt; 99％である。 </li></ol> 5。 Transuroepithelial好中球移行など言う無菌法を用いて組織培養フードで次の手順を実行します。ステップ2.7で決定されるように、コンフルエント5637細胞層を担持する透過性をサポートのみを使用してください。 RPMI-することができ、予め用意し、使用するまで5％CO 2、37℃で維持することを含む24ウェルプレート。 <ol><li> 24ウェルプレート内の一定分量1ミリリットル暖かいRPMI-ウェルあたり、透過性支持体につき3ウェルを準備します。 </li><li>透過性支持体の上部及び下部容器から培地を吸引する。 </li><li>滅菌ピンセットを使用して、ステップ5.1で作成、24ウェルプレートに透過性のサポートを転送する。ウェルからウェルに支援を転送することにより、透過性のサポートを3回洗浄します。 </li><li>細菌接種を使用する場合は、無菌の25ミリメートル深部組織培養皿に透過性支持体を反転させる。化学誘引物質（ 例えば 、N-ホルミル-Met-Leu-Phe（のfMLF）またはIL-8）を使用する場合、5.6に進む。 </li><li>ライブまたは殺さBACTを用いた実験のためにerial刺激は、60μlのRPMI-（モック感染症）、あるいは細菌接種ステップ3.5で調製された（6×10 6 CFU）を各透過性支持の頂端側に接種。皿に蓋を置き、1時間、5％CO 2、37℃でインキュベートする。 </li><li> 24ウェル低取付板によくRPMI-あたり0.6ミリリットルを追加します。透過性のサポート、ウェルあたり1を準備します。 PMN入力を列挙するために、RPMI-0.5ミリリットルを含む3つのウェルを準備します。 <ol><li>化学誘引物質は、細菌の代わりに使用されている場合は、ステップ5.6で調製した24ウェルプレートRPMI-0.6 mlに化学誘引物質を加える。 </li></ol></li><li> 24ウェル低接着プレートに右透過性をサポート。 </li><li>各透過性支持の上部貯水池（側底側）に、ステップ4.9で作成0.1ミリリットルのPMN（10 6 PMN）を追加します。直接500μL含むウェルに100μlのPMNを追加したRPMIを。 1時間、5％CO 2、37℃でインキュベートする。 </li><li>滅菌ピンセットを使用して、優しくこすりウェルの縁に透過性支持体の膜は、頂端側から追加のPMNを削除し、サポートを処分する。 </li><li>そっと千μlのピペットチップで各ウェルの底をこすることでPMNを収集し、チューブを微量遠心するPMN懸濁液を移す。 </li><li>血球計数器を用いてPMNを列挙。 </li><li>下側リザーバ内PMNの合計数を計算するには、6,000（100 nlの） は 1〜2mmでPMNの数を乗算する。データを入力PMNの割合として、または10 6の入力PMNに正規化PMNの数として報告することができる。 </li></ol>

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著者らは、開示することは何もありません。

培養された膀胱上皮細胞株および新たに単離されたヒトPMNを使用して、我々はtransuroepithelial好中球遊走のインビトロモデルを確立した。このモデルは、尿路感染症（UTI）の間に自然免疫応答、通常UPEC 9に起因する非常に一般的な細菌感染症の複雑さを分析し始めに尽力されています。膀胱、または膀胱炎の感染の間に、膀胱内腔へのPMNの補充は、細菌のクリアランス10?...

全身の細胞の動きは、多くの場合、長い距離を越え、成長と発展、創傷治癒、および免疫応答に必要です。細胞移動は複雑であり、シグナル伝達カスケードおよび細胞骨格成分の再配置を含む多くの異なるプロセスの調整を必要とする。細胞は（ケモキネシス）だけでなく、定義された化学勾配（走化性）に向かってランダムに移動することができます。多くの技術は、 インビトロで細胞遊走を研究するために開発されてきた。最も古く、最も一般的な手法、Boydenチャンバーは、化学誘引物質は下部チャンバー内に配置され、目的の細胞を上部チャンバー1内に配置された垂直2チャンバシステムで構成されています。二つのチャンバーを分離する規定サイズの孔を有する通気フィルターを横切る細胞の移動は、監視される。さらなる技術は、Zigmond室2及びダンチャンバを含む、細胞移動を研究するために開発されている3。これらの集合的なアプローチは、多くの異なる細胞型の移動に重要な洞察が得られている。 ケモキネシスおよび走化性の基本原理に問い合わせることに加えて、二チャンバーアッセイは、細胞外マトリックス成分との両方の内皮細胞および上皮細胞層を介して細胞移動の調査を促進した。他の技術上の2つのチャンバシステムの利点は、多孔質膜は、例えば、コラーゲンまたはフィブリノーゲンのようなタンパク質でコーティングすることができる、および細胞外マトリックス状の障壁を横切る細胞移動を評価することができることである。さらに、培養細胞株を増殖させ、透過性の支持体上で区別することができる。内皮障壁を横切る細胞の移動を調べるために、培養内皮細胞を播種し、透過性支持体の上部リザーバ中で増殖させる。例えば、免疫細胞などの運動性細胞は、エン全体の下池に上部貯水池と移行に追加されます生理学的な頂端から側底方向にdothelial障壁が観察される。このモデルは、血流からの免疫細胞の血管外遊出を理解する上で非常に貴重であった。移行を経内皮とは対照的に、上皮バリアを横切る細胞の移動は、典型的には、側底側から頂端方向に発生する。 インビトロでこれらのイベントをモデル化するために、研究者らは、透過性支持体の下側に培養上皮細胞を播種し、成長する。運動性の細胞は側底から頂端方向を表す、上皮バリアを横切る上側リザーバおよび遊走に追加され、監視される。経上皮の移行のようなモデルが大幅に粘膜表面での炎症反応、特に肺のものと、4,5腸の我々の理解に貢献した。 対照的に、尿路の上皮を介して免疫細胞の輸送はそれほど注目されている。私たちのunderstandiを促進するために膀胱上皮バリア6渡って動き、（好中球、PMN）NG尿路病原性大腸菌 （UPEC）による感染時の尿路内の自然免疫応答のため、我々は多形核白血球の調査を可能にするin vitroアッセイ、transuroepithelial好中球遊走アッセイを開発-8。経上皮遊走の他の二チャンバーモデルと同様に、培養されたヒト膀胱上皮細胞は、透過性支持体の下側に増殖させ、コンフルエント上皮層を形成している。静脈血から単離されたヒト好中球は、上皮層の側底側に適用され、生理学的に関連する側底から頂端方向に上皮を横切る遊走は、E.の異なる株による感染に応答して定量化される大腸菌や化学誘引分子の存在。多くの研究は、追加の細胞型の非存在下での好中球の移動、ケモキネシスおよび走化性の両方に焦点を当てていた。それは考慮感染の間、膀胱上皮細胞と免疫細胞との間の複雑な相互作用を要するようtransuroepithelial好中球遊走アッセイは有利である。この扱いやすいin vitroモデルは、尿路上皮表面における免疫応答の詳細な調査を許可する可能性を秘めている。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Transwell Inserts (i.e.&#160;permeable supports)</td>
			<td>Corning</td>
			<td>3472</td>
			<td>6.5 mm, 3 &#956;m pore, polyester membrane insert</td>
		</tr>
		<tr>
			<td>BD Vacutainer Blood Collection Tubes</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>366480</td>
			<td>16 mm x 100 mm x 10 ml green cap tubes containing sodium heparin</td>
		</tr>
		<tr>
			<td>BD Safety-Lok Blood Collection and Infusion Set</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>367281</td>
			<td>21 G x 0.75 in needle x 12 in tubing</td>
		</tr>
		<tr>
			<td>BD Vacutainer one-use, nonstackable holder</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>364815</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>D4876</td>
			<td>From Leuconostoc mesenteroides</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS density centrifugation solution</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment Plates</td>
			<td>Corning</td>
			<td>3473</td>
			<td>24-well, clear flat bottom</td>
		</tr>
		<tr>
			<td>N-Formyl-Met-Leu-Phe (fMLF)</td>
			<td>Sigma-Aldrich</td>
			<td>F3506</td>
			<td>Reconstituted in DMSO to 10 mM</td>
		</tr>
		<tr>
			<td>Recombinant Human CXCL8/IL-8</td>
			<td>R&#38;D Systems</td>
			<td>208-IL</td>
			<td>Reconstituted in PBS to 100 &#956;g/ml</td>
		</tr>
	</tbody>
</table>

quantitative assessment of human neutrophil migration across a cultured bladder epithelium

炎症部位への周囲からの免疫細胞の動員は、任意の粘膜表面での自然免疫応答において重要なステップです。膀胱の感染の間に、多形核白血球（PMN、好中球）は、血流から移動した膀胱上皮を横断する。好中球応答がない場合に感染を解決しないと、膀胱防御にPMNの重要性を示している。膀胱上皮、尿路病原性大腸菌 （UPEC）、尿路感染症（UTI）の大部分の原因物質の定着を促進するために、部分的に定義された様々な機構を使用して急性炎症反応を弱める。さらに、ホストおよび細菌病原体の間の相互作用を調べるために、我々はUPECに対する自然免疫応答のこの態様のインビトロモデルを開発した。 transuroepithelial好中球遊走アッセイでは、Boydenチャンバーのバリエーション、培養された膀胱epithelial細胞は透過性支持体の下側に密集するまで成長させる。 PMNは、ヒト静脈血から単離され、膀胱上皮細胞層の側底側に適用される。細菌感染または化学誘引物質の分子に反応して、生理的に適切な基底外側への頂端方向を表すPMNの移行は、血球計数器を使用して列挙されている。このモデルは、UPECおよび真核細胞間の相互作用を調査するために、ならびにPMNによる膀胱上皮のトラバーサルのための分子の要件を調べるために使用することができる。 transuroepithelial好中球遊走モデルは、膀胱内のUPECへの初期炎症反応の我々の理解を促進します。

transuroepithelial好中球遊走アッセイは、種々の刺激（ 図1B）に対応して培養し、膀胱上皮細胞層全体の人間のPMNの移行の定量的評価が可能になります。プロトコルは単純であるが、PMN遊走に影響を及ぼし、その結果、このアッセイの再現性に影響を与えることができる変数の数がある。技術的、生物学的反復間の変動を減らすために透過性をサポートし、PMNの準備中な処置を行う?...

Watch this Scientific Journal Video about Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium at JoVE.com

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

私たちは、尿路病原性大腸菌で膀胱の感染中の急性の炎症反応の重要な要素を模倣したin vitroモデルを開発しました。 transuroepithelial好中球遊走アッセイは、細菌感染や化学誘引物質に反応して、透過性の支持体上で培養、膀胱上皮間でヒト好中球の遊走の定量的評価が可能になります。

培養された膀胱上皮を越えるヒト好中球の移行の定量的評価

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

quantitative-assessment-human-neutrophil-migration-across-cultured

Research

JoVE Journal

Immunology and Infection

15.0K ビュー.  Washington University School of Medicine. 私たちは、尿路病原性大腸菌で膀胱の感染中の急性の炎症反応の重要な要素を模倣したin vitroモデルを開発しました。 transuroepithelial好中球遊走アッセイは、細菌感染や化学誘引物質に反応して、透過性の支持体上で培養、膀胱上皮間でヒト好中球の遊走の定量的評価が可能になります。 

ビデオ: Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

拡大、精製、及びヒト末梢血NK細胞の機能的評価

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

免疫・感染学

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

生体内ビデオ顕微鏡による組織内好中球腔内クロールを追跡、経内皮移動と走

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Phagokineticトラック運動アッセイによる細胞遊走の定量的評価

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

定量的な<em&gt;インビトロ</em静的な条件の下で活性化された初代ヒト微小血管内皮細胞への好中球の接着を測定するために&gt;アッセイ

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

インビトロ 病原体誘導性好中球トランス上皮移動を評価するコーカルチャーアッセイ

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

ヒト好フローチャンバー接着アッセイ

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

ヒト好中球由来の巨大食細胞の新規亜集団の開発および同定<em&gt;インビトロ</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

体外蛍光顕微鏡による犬の好中球細胞外トラップ形成: ダイナミックな定量的分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

排ビボ移行システムにおけるリンパ球移動の評価

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

標準化腸ループモデルを用いたマウスにおける腸透過性と好中球経上皮移動の機能評価

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...