この作品は、カナダ衛生研究所（CIHR、MOP - 86749）から研究助成金によってサポートされていました。 L. LiuはCIHR新奨励賞（MSH - 95374）の受信者です。

1。アガロースゲルでの誘引物質の調製<ol><li>ピペット10 50 - mLコニカルチューブに2 × PBSの添加、および熱水を入れたビーカーに入れておくことによりチューブのウォームアップを行います。 </li><li>ピペット10の蒸留水を加えて、別の50 mLコニカルチューブに0.4 Gアガロースの粉末を加える（少し緩め、キャップ付き）、混合物を加熱（700ワットの電子レンジで約1分間）電子レンジのちょうど沸騰するまで。 </li><li>アガロース溶液のチューブ、渦混合溶液に加温し、2 × PBSを追加し、お湯のビーカーに、冷えないように保管してください。 </li><li>マイクロピペットを用いて吸引しても3μlのインドのインクと混合を含む1.5 mLのエッペンドルフチュー​​ブの蓋に、マイクロピペット走化性溶液（例えば、CXCケモカインMIP - 2または1 mM WKYMVmの12μlの0.5μgの10μlのは）（避ける空気の泡）。 </li><li>蓋に200μlのピペットチップおよびアガロース溶液（42 ° C）のマイクロピペット110μlの先端をカットし、すぐに別のピペットチップを（空気の泡を避けるため）を使用してよく混ぜる。 </li><li> +4化学誘引物質含有ゲル℃にて保存してください。 </li></ol> 2。生体顕微鏡検査のための精巣挙筋の調製（図1） <ol><li> 10 mg / kgのキシラジン、200 mg / kgの塩酸ケタミンの混合物の腹腔内注射による成熟雄性マウスを麻酔。 </li><li>右外頸静脈と電気かみそりと陰嚢の前面を覆う領域を剃る。麻酔後、麻酔マウスに特別な注意とケアを提供することが重要です。ヒートランプは低体温症からマウスを防止するために使用されることがあります。マウスは、痛みの反射のない状態にしてください。 </li><li>水平方向の切開を行う、100 U / mLのヘパリンの生理食塩水で満たされたPE - 10チューブを使用して頚静脈を見つけて、カテーテルを挿入する。必要な時に頸静脈のカテーテル挿入は、追加の麻酔薬や薬の投与のために必要です。 </li><li>マウスは、ホームメイドの精巣挙筋板（図1）に表向きに横たわっていると臍テープでマウスの後ろ足を修正。 </li><li>精巣挙筋とマウスの体を暖かく保つために37℃の水循環にボードを接続する。 </li></ol>注：2.6から2.14までのすべての手順は非常に穏やかに5を実行する必要があります。 <ol start="6"><li>左精巣挙筋を露出するために陰嚢皮膚に切開を加えます。慎重に関連付けられている筋膜から筋肉を細かく分析。 </li><li> ° Cに加温した蠕動ポンプを用いて（131.9のNaCl、4.7 KClを、1.2のMgSO 4、20mMの中のNaHCO 3を 、液、pH7.4）生理食塩水重炭酸緩衝化37で精巣挙筋の表面かん流する。 </li><li>精巣挙筋板の透明な観覧のガラスの台座の上にそれを保持するために精巣挙筋の遠位端に4から0縫合糸を結びます。 </li><li>縦方向に精巣挙筋を焼灼する。 4から0縫合糸で、筋肉のフラットを保持し、台座のエッジに沿って固定します。基本となる筋肉から睾丸と精巣上体を分離し、腹腔内にそれらを移動する。 </li><li> 37 ° Cに加温した灌流バッファ付き〜0.6 ml /分で筋肉を表面かん流すると22 × 22 mmのカバーガラスで露出筋肉をカバー。 </li><li> 、顕微鏡のステージ上で精巣挙筋のボードを置き、顕微鏡で筋肉を調べ、適切なpostcapillary静脈を（ストレートおよび非分岐であり、通常のせん断速度25〜40ミクロ​​ンの直径を有する細静脈を選択して）検索して、ビデオカメラを調整する細静脈は、テレビモニターの左右どちらかの端に垂直位置に可視化できるようにする。 </li><li> 30分の平衡した後、ビデオレコーダーを使用してベースラインの制御データとして、5分間選択postcapillary細静脈のビデオ画像を記録する。 </li><li>灌流を停止し、筋肉でカバーを取り外します。 </li><li> 350μmであるから、事前に選択されたエリアでの精巣挙筋の表面上に約1 mmの3サイズの化学誘引物質を含むゲルを置き、平行観測postcapillary細静脈に、代わりにゲルを保持するためにカバースリップを追加し、非常に筋肉組織を表面かん流する徐々に解放され、ゲルから形成される走化性因子の勾配の確立を可能にするために遅い速度（≤10μL/分）。 </li><li>化学誘引物質を含むゲルの添加後90分間のビデオ画像を記録する。録音中は、静脈内および筋肉組織の白血球をtransmigratingとchemotaxing、クロール、付着に関する顕微鏡のフォーカスを調整してください。 </li><li>実験後、解析のためのコンピュータにビデオファイルをインポートします。 </li></ol> 3。 ImageJを用いた細胞の追跡<ol><li>コンピュータ上で、抽出して、AVI形式（例えば、AVIファイルにDVDビデオを変換する無料のコンピュータソフトウェアのbitRipperを使用）にビデオを変換します。 </li><li>時間が経過した動画を生成するためのビデオ編集ソフトウェアを使用してください。例えば、時間が経過した動画を（1 / 512または1024分の1の時間経過のためにWindowsムービーメーカーを使用してくださいオリジナルの、リアルタイムのビデオからT 30 - fpsレート）。 DV - AVI形式に圧縮されていない時間が経過した動画を変換して保存。 </li><li>同じ顕微鏡の設定でキャリブレーションマイクロメートルの画像を記録し、コンピュータに画像をインポートし、ImageJので画像を開きます。 ImageJのでは、総ピクセルが画面の左上（例えば、720 × 480ピクセル）に表示されます。 X軸とY軸（例えば、200 × 150μm）の両方で画面の大きさを測定します。このことから、ミクロンあたりのピクセル（例えば、X = 200分の720 = 3.6ピクセル/μmの、およびy = 150分の480 = 3.2ピクセル/μm）の数を計算する。 </li><li>再び映画、オープンImageJをインポートするには、&quot;ファイル - インポート - 使用して、QuickTimeムービープラグイン&quot;をクリックして、分析するムービーを選択し、&quot;QTムービーの開閉&quot;のインタフェースで&quot;OK&quot;をクリックします。 </li><li>細胞を追跡するために、&quot;プラグイン - マニュアルトラッキング&quot;をクリックしてください。開始トラッキングの前に下部にあるフィールドに関連する情報を入力してください。簡単に言えば、 <ol><li>時間間隔（秒）= fold-time-lapse/30（例えば、1020 ×タイムラプス= 34秒/フレームの間隔を1020から1030になる）。 </li><li> x / yのキャリブレーション=マイクロメートルの画像のキャリブレーションを使用してμm/ピクセル測定。 </li><li> Z校正= 0（マウス精巣挙筋は組織や細胞のクロールと移行の非常に薄い層であるとして明視野透視下で約2Dです）。 </li><li>センタリング= 1の正方形のサイズを検索します。 </li><li>ドットサイズ/線幅/フォントサイズ：必要に応じてそれらを調整することができます。 </li></ol></li><li>基準点として、安定かつ明瞭なポイントを選択します。この基準点は、全実験を通じて変化と安定している明確な、小さな構造のポイントになります。最後のフレームに最初から基準点を追跡し、&quot;終了トラック&quot;をクリックして&quot;トラックを追加&quot;をクリックします。データは自動的に結果表に表示されます。 </li><li>好中球をクロールし、移行を追跡一つずつ：クリックすると、各フレームにおけるその消失する組織において、その外観から細胞を追跡し、Microsoft Excelでの結果を終了して保存するには&quot;終了トラック&quot;をクリックして&quot;トラックを追加&quot;。 </li></ol> 4。好中球リクルートパラメータの解析<ol><li> Microsoft Excelで結果のファイルを開き、（分析に基準点の変更を行う）、データを分析する。 </li><li>腔内クロール<ol><li>距離をクロール：合計距離はセルが最適な移住地（μm）に初期接着部位から内腔にクロール。 </li><li>クロール速度：クロールの距離/時間（μm/分）。 </li></ol></li><li>経内皮移動<ol><li>移住の時間：セル全体の細胞体は、細静脈の外側だけであるとは細胞体が内腔（分または秒）で見られない時の時間に内皮を越え転生するために開始時間から。 </li><li>剥離は時間：時から全体の細胞体は単なる静脈外にある（すぐに移住後の）時点にセルが細静脈（尾部が縮んだ状態）（分または秒）との接触を失う。 </li></ol></li><li>組織の走化性<ol><li>移行距離：距離の和開始点から組織のマイグレーション（μm）のエンドポイントへの細胞移動。 </li><li>移行の速度：組織/時間の移動距離（μm/分）。 </li><li>走化性の距離：距離のセルの合計は、組織におけるx軸（μm）で移行。 </li><li>走化性の速度：走化性の距離/時間（μm/分）。 </li><li>走化性指数：組織の移動距離で走化性の距離を分割する比率。 </li></ol></li></ol> 5。代表的な結果： bightfield生体顕微鏡が白血球 - 内皮細胞相互作用の研究のために使用されており、好中球のために、必ずしもではないかもしれないが、我々は組織学的研究によって、好中球走化性処理精巣挙筋の動員細胞の95％以上が実際に好中球であったことが確認。このレポートでは、好中球選択的誘引物質を使用して、我々はin vivoでの好中球の動員を追跡する手順を紹介します。具体的には、トラッキング好中球腔内クロール、経内皮移動、およびタイムラプス生体内ビデオ顕微鏡とImageJを用いて麻酔したマウスの精巣挙筋の組織における走化性のプロトコルを記述する。精巣挙筋上に化学誘引物質含有アガロースゲルは、徐々に化学誘引物質を放出し、走化性勾配が組織に確立することができます。好中球の走化性因子は、マウスの精巣挙筋postcapillary細静脈における好中球 - 内皮細胞の相互作用を誘導する。全体の実験では、テレビモニターにカラービデオカメラで投影し、ビデオレコーダで記録されたビデオイメージを持つ正立明生体顕微鏡下で可視化です。我々は、好中球を決定アガロースゲル（図2）で調製した化学誘引物質MIP - 2とWKYMVmを好中球への応答における筋肉組織の内腔クロール、経内皮移動と移行し、走化性。我々は、MIP - 2（0.5μMで）とWKYMVmが（0.1 mmで）ほぼで同等の時間の長さは、好中球の遊走および筋肉組織における走化性のための静脈から同じような速度、好中球内皮遊走および剥離時に好中球腔内クロールを引き出したことがわかった同じ速度および同じような好中球の走化性のインデックスを持つ（P&gt; 0.05、スチューデントt検定）。 <img src="/files/ftp_upload/3296/3296fig1.jpg" alt="図1" /> 図1。生体顕微鏡システムの概略図。マウス精巣挙筋は、顕微鏡ステージ上で精巣挙筋板の透明な観覧の台座の上に体外と37℃に加温した重炭酸塩緩衝食塩水で灌流される。正立顕微鏡は、明視野生体顕微鏡用CCDカラービデオカメラと接続されています。モノクロ深い冷却CCDデジタルカメラも蛍光生体顕微鏡用顕微鏡のポートに接続され、イメージはそこから直接コンピュータによって処理されます。 <img src="/files/ftp_upload/3296/3296fig2.jpg" alt="図2" /> 明生体顕微鏡の図2。好中球動員のパラメータ。好中球の動員は、アガロースゲルの準備のMIP - 2またはWKYMVmがpostcapillary細静脈に隣接350μmであるが置かれた好中球の走化性因子が徐々に放出によって誘発された。時間経過ビデオデータは、実験のリアルタイムのビデオ録画を処理した後、ImageJのによって分析した。精巣挙筋（D）における好中球内腔クロール（）、移住の時間と剥離の時間（B）、組織の移動速度と走化性の速度（C）、および走化性の指数は、MIP - 2またはWKYMVmアガロースゲル上での投与後に測定したC57BL / 6マウスの精巣挙筋（n = 3の、＃追跡セルの= 22（AとBで）、27（CおよびDで）それぞれMIP - 2の、と= 26（AとB）、44（ WKYMVmため、それぞれCとD）で）。

<ol>
	<li>Liu, L., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+leukocyte+recruitment:+organ-specific+mechanisms+of+action.">Molecular mechanisms of leukocyte recruitment: organ-specific mechanisms of action.</a> Thromb. Haemost. 89, 213-220 (2003).</li><li>Petri, B., Phillipson, M., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+leukocyte+recruitment:+an+in+vivo+perspective.">The physiology of leukocyte recruitment: an in vivo perspective.</a> J. Immunol. 180, 6439-6446 (2008).</li><li>Phillipson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraluminal+crawling+of+neutrophils+to+emigration+sites:+a+molecularly+distinct+process+from+adhesion+in+the+recruitment+cascade.">Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade.</a> J. Exp. Med. 203, 2569-2575 (2006).</li><li>Wong, C. H., Heit, B., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulators+of+leucocyte+chemotaxis+during+inflammation.">Molecular regulators of leucocyte chemotaxis during inflammation.</a> Cardiovasc. Res. 86, 183-191 (2010).</li><li>Gavins, F. N., Chatterjee, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+for+the+study+of+mouse+microcirculation+in+anti-inflammatory+drug+research:+focus+on+the+mesentery+and+cremaster+preparations.">Intravital microscopy for the study of mouse microcirculation in anti-inflammatory drug research: focus on the mesentery and cremaster preparations.</a> J. Pharmacol. Toxicol. Methods. 49, 1-14 (2004).</li><li>Bullen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+imaging+techniques+for+drug+discovery.">Microscopic imaging techniques for drug discovery.</a> Nat. Rev. Drug Discov. 7, 54-67 (2008).</li><li>Hazelwood, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entering+the+Portal:+Understanding+the+Digital+Image+Recorded+Through+a+Microscope.">Entering the Portal: Understanding the Digital Image Recorded Through a Microscope.</a> Imaging Cellular and Molecular Biological Functions. , 3-43 (2007).</li><li>Hickey, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-selectin+facilitates+emigration+and+extravascular+locomotion+of+leukocytes+during+acute+inflammatory+responses+in+vivo.">L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo.</a> J. Immunol. 165, 7164-7170 (2000).</li><li>Cara, D. C., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+as+a+tool+for+studying+recruitment+and+chemotaxis.">Intravital microscopy as a tool for studying recruitment and chemotaxis.</a> Methods Mol. Biol. 239, 123-132 (2004).</li><li>Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LSP1+is+an+endothelial+gatekeeper+of+leukocyte+transendothelial+migration.">LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration.</a> J. Exp. Med. 201, 409-418 (2005).</li><li>Heit, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+accelerates,+but+is+not+required+for,+neutrophil+chemotaxis+to+fMLP.">PI3K accelerates, but is not required for, neutrophil chemotaxis to fMLP.</a> J. Cell Sci. 121, 205-214 (2008).</li></ol>

利害の衝突は宣言されません。

生体顕微鏡検査は、炎症時に白血球動員の細胞および分子メカニズムを明らかにするための不可欠なツールです。このような精巣挙筋および腸間膜としてtransluscent組織の微小血管系における白血球-内皮細胞の相互作用を測定するための定量的可視化は、技術1,5のアプリケーションのためのゴールドスタンダードのまま。従来の明視野生体顕微鏡は、多くのユニークな技術的特徴を有?...

表：材料、具体的な試薬および器具<table border="1"><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ポリエチレンチューブ、PE10 </td><td>ベクトンディッキンソン</td><td> 427401 </td><td> ID 0.28ミリメートル×外径0.61ミリメートル</td></tr><tr><td>インドインク</td><td>スピードボールアート</td><td>スーパーブラック</td><td> 100％のカーボンブラック顔料、無色素</td></tr><tr><td>焼灼</td><td>アーロンメディカル</td><td> AA03 </td><td></td></tr><tr><td>キシラジン</td><td>バイエルヘルスケア社、バイエル（株） </td><td> DIN 02169592 </td><td></td></tr><tr><td>塩酸ケタミン</td><td> Bionicheアニマルヘルスカナダ、（株） </td><td> DIN 01989529 </td><td></td></tr><tr><td>マウス組換え型MIP - 2 </td><td> R＆Dシステムズ</td><td> 452 - M2 </td><td></td></tr><tr><td> WKYMVm </td><td>フェニックスファーマシューティカルズ</td><td> 072から12 </td><td></td></tr><tr><td>アガロース</td><td>インビトロジェン</td><td> 15510-027 </td><td>超高純度</td></tr><tr><td>ヘパリン</td><td>シグマ</td><td> H - 3393 </td><td></td></tr><tr><td>正立顕微鏡</td><td>オリンポス</td><td> BX61WI </td><td></td></tr><tr><td> 3CCDカラービデオカメラ</td><td> SONY </td><td> DXC - 990 </td><td></td></tr><tr><td> HD - DVDビデオレコーダー</td><td> LG電子（株） </td><td> RH398H - M </td><td></td></tr><tr><td>テレビモニター</td><td> LG電子（株） </td><td> 22LG30 </td><td></td></tr><tr><td>水循環装置</td><td>サーモサイエンティフィック</td><td> HAAKEのDC10 </td><td></td></tr><tr><td>蠕動ポンプ</td><td>ギルソン;ファルマシア</td><td>ジルソンMINIPULS 3;ファルマシアP - 3 </td><td></td></tr><tr><td>精巣挙筋板</td><td>サスカチュワン州の大学</td><td></td><td>自家製の</td></tr></table>

tracking neutrophil intraluminal crawling, transendothelial migration and chemotaxis in tissue by intravital video microscopy

血流から炎症組織への循環白血球の動員は、炎症の1,2の重要かつ複雑なプロセスです。炎症組織のpostcapillary細静脈では、細静脈壁の内腔表面に最初にテザーとロールを白血球。内皮に​​白血球の逮捕を圧延し、細静脈の表面のケモカインまたは他の化学誘引物質に応答して、強固な接着を受ける。多くの接着白血球が接着の初期のサイトから内皮の接合部の血管外漏出のサイトに移転、プロセスが3クロール腔内と呼ばれる。クロールに続いて、白血球は内皮上を移動（移住）と誘引物質のソース（走化性）4に向かって血管外組織に移行する。生体顕微鏡は、 生体内で白血球-内皮細胞の相互作用を可視化し、白血球動員2,5の細胞および分子メカニズムを明らかにするための強力なツールです。このレポートでは、我々は、好中球走化性因子の勾配に応答してマウス精巣挙筋の好中球動員の詳細なプロセスを視覚化し、決定するために明生体顕微鏡を用いての包括的な説明を提供しています。好中球動員、好中球走化MIP - 2（CXCL2、CXCケモカイン）またはWKYMVm（TRP -リジン-チロシン- VAL - D - Metは、合成アナログを含むアガロースゲルの小片（〜1 mmの3サイズ）を誘導する細菌ペプチドの）観測postcapillary細静脈に隣接した筋肉組織に配置されます。時間経過したビデオの撮影とコンピューターソフトImageJのと、好中球内皮上腔クロール、好中球内皮遊走や組織におけるマイグレーションと走化性を可視化し、追跡されます。このプロトコルは、管腔内クロール速度、移住の時、剥離の時間、移動速度、組織における走化性の速度と走化性指数などの多くの好中球動員のパラメータの信頼性と定量分析が可能になります。我々は、このプロトコルを使用して、これらの好中球動員のパラメータが安定的に決定され、単一の細胞移動が便利にin vivoで追跡することができることを示している。

Watch this Scientific Journal Video about Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy at JoVE.com

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

我々は、in vivoでの好中球走化性因子のソースに対応して好中球動員中に動的な好中球 - 内皮細胞の相互作用を測定するための明生体顕微鏡検査のプロトコルを記述する。好中球内腔クロール、マウス精巣挙筋の組織の経内皮移動と走化性は、時間の経過したビデオ撮影すると、ImageJで追跡と視覚化されます。

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 ...

tracking-neutrophil-intraluminal-crawling-transendothelial-migration

Research

JoVE Journal

Immunology and Infection

19.5K ビュー.  University of Saskatchewan. 我々は、in vivoでの好中球走化性因子のソースに対応して好中球動員中に動的な好中球 - 内皮細胞の相互作用を測定するための明生体顕微鏡検査のプロトコルを記述する。好中球内腔クロール、マウス精巣挙筋の組織の経内皮移動と走化性は、時間の経過したビデオ撮影すると、ImageJで追跡と視覚化されます。

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

好中球細胞外トラップ：それらを生成して可視化する方法

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

免疫・感染学

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

鼠径リンパ節の生体顕微鏡

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

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 ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

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. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

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. ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

胚中心の高解像度生体内ストライプ照明顕微鏡

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

リアルタイムで麻酔したマウスに生体顕微鏡により腸間膜静脈に好中球および単球のイメージング

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

IL-1βプロモーター駆動のDsRedレポーターマウスを用いた好中球プライミングの生体内イメージング

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

血液滴からの迅速な好中球走化性解析のためのオールオンチップ法

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

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 ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

イメージング 2 光子生体顕微鏡を用いたインフルエンザ ウイルス感染症の中に気管粘膜の細胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...