この作品は、生物医学研究とNIH RO1 AI083426 - 01（FW）の卓越性のためのスミスファミリー賞によって資金を供給された。パトリックMaschmeyerとメラニーフラックは、ベーリンガーインゲルハイム財団からの博士課程の奨学金によってサポートされています。

C57BL / 6マウスは〜20週齢以上で使用してください。 25〜30グラムの重さ、雄マウスの使用が推奨されています。星状細胞の収量は、細胞の分離を星状に先立って2ヶ月のためにマウスにビタミンAに富む食事を供給することにより増加することができます。 Balb / cマウスからの肝星細胞の収率がかなり高いのに対し、約2 × 10 5肝星細胞は、一つC57BL / 6マウスの肝臓から精製することができる。以下のプロトコルは、5匹のマウスに調整されます。動物のケアと実験が承認された動物実験に基づき行われ、委員会のプロトコルを使用していた。 （1）マウスの肝臓の in situ灌流の消化酵素で<ol><li> 37℃の水浴中でSC1バッファーと酵素の灌流液を温める。 </li><li>蠕動ポンプのシリコンチューブにセット翼の注入をマウントします。 </li><li>肝臓の現場灌流のために使用される流量である6.5ml/min、の層流を得るためにPBSでポンプのキャリブレーション。 SC1溶液をシリコンチューブを平衡化する。 </li><li>ケタミン（90 mg / kg体重）及びキシラジン（10 mg / kg）のソリューションと深いnarcotizationを確保するために反射の消失のためのテストの腹腔内注射によってマウスを麻酔。 </li><li>適当な塩基での仰臥位でマウスを修正。 </li><li>腹部皮膚に縦切開を行い、腹膜を露出させるハサミとピンセットを使用してください。 </li><li>慎重に門脈を露出させるために腹膜を開き、腹腔の動物の左側に腸を動かす。 </li><li>門脈に設定された注入のカニューレを挿入します。このステップでは、実体顕微鏡の使用が推奨されます。 </li><li> 30ミリリットルSC1液と肝臓の灌流を開始します。フローの開始直後に下大静脈を開きます。肝臓の成功したフラッシュは肝臓の組織の色の消失によって示されます。 </li><li> 30ミリリットルプロナーゼE溶液を用いて肝臓を灌流。成功した消化されると、肝臓の葉が膨張します、と小葉は、カプセルを介して異なる表示されます。 </li><li> 30ミリリットルコラゲナーゼP溶液で肝臓を灌流。この時点で肝臓はその形を失い、弛緩と非晶質に見えるしている。 </li><li>慎重に横隔膜と周囲の臓器から肝臓を分離し、氷上で70ミリリットルSC2ソリューションに格納します。 </li><li>追加のマウスのための手順を繰り返します。 </li></ol> 2。マウスの肝臓の in vitro消化において それ以後のすべての作業手順は、層流フードで無菌条件下で行う必要があります。 <ol><li>良く切れるはさみを使用して約2x2x2mm 3の部分に肝臓を切り取ります。 </li><li>プロナーゼE -コラゲナーゼP溶液50mlのある肝臓の作品を含む70ミリリットルSC2のサスペンションを組み合わせるとDNase I溶液の1mlを加える。 </li><li> 37℃20分のための肝臓を消化° Cで撹拌しながら。 </li></ol> 3。密度勾配遠心<ol><li> 6 50ミリリットルのファルコンチューブに70μmセルストレーナーを通して細胞懸濁液を濾過し、SC2バッファーで50ミリリットルまで追加。 600グラムと4℃で10分間遠心</li><li>注意深く上清の40ミリリットルを吸引し、各チューブに150μLDNase I溶液を追加し、細胞を懸濁します。 </li><li> 4 50ミリリットルファルコンチューブにプール細胞懸濁液をし、600グラムと4℃で10分間GBSS - B、遠心機で洗う℃に</li><li>慎重にペレットと各チューブへ追加150μLDNase I溶液を乱すことなく、可能な限り上清のを吸引除去する。チューブあたり10ミリリットルGBSS - Bの細胞ペレットを再懸濁します。 </li><li>プールは2 50ミリリットルファルコンチューブに細胞や試験管当たり36ミリリットルの合計体積にGBSS - Bを追加します。各チューブに14ミリリットルナイコデンツのソリューションを加え、よく混ぜる。 </li><li>合計で10本で、その結果、一12ミリリットル勾配遠心チューブに細胞懸濁液10mlのを転送する。優しくチューブあたり1.5ミリリットルGBSS - Bで細胞懸濁液を重ねます。 </li><li> 1500グラムと4℃で15分のための勾配を遠心° Cブレーキなし。星状細胞は白い輪のような相間で発見されているのに対し、その後、肝細胞がチューブの底にペレット化されます。 </li><li>慎重に星状細胞を含む中間相を収穫し、GBSS - Bでそれらを洗って、10分間遠心するを600グラムと4℃ </li><li>上清を吸引除去し、10％熱不活性化FBS、1％ペニシリン/ストレプトマイシン、2mMのL -グルタミン、1mMのピルビン酸ナトリウム、及び10mMのHEPESを添加した20ミリリットルDMEMで細胞を懸濁します。 2 × 10 4細胞/ cm 2の濃度で組織培養フラスコに細胞を移す。 37℃、5％CO 2で細胞をインキュベートする。 </li><li>とすぐに肝星細胞は死細胞と細胞残渣を洗い流すために（約2時間の準備の後に）接着なので、メディアを変更します。 </li><li>翌日、星状細胞でsは核周囲ビタミンAを格納する脂質小胞との特徴的な星型の形態を開発する必要があります。 </li><li>その後の実験では、肝星細胞は容易にそのようなAccutase、などなど、軽度の酵素的ソリューションを使用して、非コーティングプラスチック表面から切り離すことができます。 </li></ol> 4。代表的な結果： 提供するプロトコルを使用して肝星細胞の調製後、孤立した個体群の純度は、3等星のような形としてこの細胞型の主な特性は、核周囲の脂質滴、およびグリア線維性酸性タンパク質（GFAPの発現を考慮してテストすることができます）。特徴的な細胞分離後の肝星細胞の2Hの外観だけでなく、in vitro培養の1日目及び3のための代表的な写真を図1に示されている。図2は、細胞の単離後3日間培養された肝星細胞におけるGFAPのための代表的な免疫蛍光染色を示す。肝星細胞は、in vitro培養時の筋線維芽細胞へと分化する。それらの活性化星細胞の特徴特徴は、α平滑筋アクチン（αSMA）の式です。図3は、in vitro培養の7日目に活性化星細胞のアスマとミオシンIIAのための免疫蛍光染色を示す。 <img src="/files/ftp_upload/2710/2710fig1.jpg" alt="図1" /> 図1肝星細胞の特徴的な形態。星状細胞は、提供されるプロトコルを使用してマウスの肝臓から単離された。画像は、星状細胞の細胞単離後2H（A、B）、ならびに1日目（C）とin vitro培養の3日目（D）上を表しています。肝星細胞は、核周辺部位での脂質小胞の高い量を示し、in vitro培養の最初の日（倍率200倍）の間に彼らの独特のアストラル様形態を獲得する。 <img src="/files/ftp_upload/2710/2710fig2.jpg" alt="図2" /> 図2肝星細胞は、特に肝臓でのGFAPを発現している。星細胞は、in vitro培養の3日目に単離され、immunofluorescently GFAP（赤）で染色した。細胞核は青色（ヘキスト染色）に示されている。 <img src="/files/ftp_upload/2710/2710fig3.jpg" alt="図3" /> 図3。肝星細胞が筋線維芽細胞に分化する。 C57BL / 6マウスから単離した肝星細胞は7日間培養した。その後、彼らは、チャンバースライドに移し、そしてアスマ（赤で表示）とミオシンIIA（緑で示される）のために染色した。細胞核はヘキスト（青色）で対比染色した。 <table border="1"><tbody><tr><td colspan="2"> SC1バッファ </td></tr><tr><td> EGTA </td><td> 190mg </td></tr><tr><td>グルコース</td><td> 900mg </td></tr><tr><td> HEPES </td><td> 1Mストック溶液10mlを</td></tr><tr><td>塩化カリウム</td><td> 400mgを</td></tr><tr><td>のNa 2 HPO 4 × 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaClの</td><td> 8グラム</td></tr><tr><td>のNaH 2 PO 4 × H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3の </td><td> 350mg </td></tr><tr><td>フェノールレッド</td><td> 6mgを</td></tr><tr><td>のdH 2 O </td><td> 1リットルに一杯に</td></tr><tr><td colspan="2"> SC2バッファ </td></tr><tr><td>塩化カルシウム2 × 2H 2 O </td><td> 560mg </td></tr><tr><td> HEPES </td><td> 1Mストック溶液10mlの</td></tr><tr><td>塩化カリウム</td><td> 400mgを</td></tr><tr><td>のNa 2 HPO 4 × 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaClの</td><td> 8グラム</td></tr><tr><td>のNaH 2 PO 4 × H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3の </td><td> 350mg </td></tr><tr><td>フェノールレッド</td><td> 6mgを</td></tr><tr><td>のdH 2 O </td><td> 1リットルに一杯に</td></tr><tr><td colspan="2"> GBSS -バッファ </td></tr><tr><td>塩化カリウム</td><td> 370mg </td></tr><tr><td>塩化カルシウム2 × 2H 2 O </td><td> 225mg </td></tr><tr><td>グルコース</td><td> 991mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mgを</td></tr><tr><td>のMgCl 2 × 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4を 365 H 2 O </td><td> 70mg </td></tr><tr><td>のNa 2 HPO 4 × 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaHCO 3の </td><td> 227mg </td></tr><tr><td>フェノールレッド</td><td> 6mgを</td></tr><tr><td>のdH 2 O </td><td> 1リットルに一杯に</td></tr><tr><td colspan="2">GBSS - Bバッファ </td></tr><tr><td>塩化カルシウム2 × 2H 2 O </td><td> 225mg </td></tr><tr><td>グルコース</td><td> 991mg </td></tr><tr><td>塩化カリウム</td><td> 370mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mgを</td></tr><tr><td>のMgCl 2 × 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4を 365 H 2 O </td><td> 70mg </td></tr><tr><td>のNa 2 HPO 4 × 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaClの</td><td> 8グラム</td></tr><tr><td> NaHCO 3の </td><td> 227mg </td></tr><tr><td>フェノールレッド</td><td> 6mgを</td></tr><tr><td>のdH 2 O </td><td> 1リットルに一杯に</td></tr><tr><td colspan="2"> プロナーゼEの灌流液 </td></tr><tr><td>プロナーゼE </td><td> 100mgの（4000 PU / MG分） </td></tr><tr><td> SC2バッファ</td><td> 200ミリリットル</td></tr><tr><td colspan="2"> コラゲナーゼPの灌流液 </td></tr><tr><td>コラゲナーゼP </td><td> 85mg（1.78 U / mgのlyo） </td></tr><tr><td> SC2バッファ</td><td> 200ミリリットル</td></tr><tr><td colspan="2"> プロナーゼE -コラゲナーゼPソリューション </td></tr><tr><td>プロナーゼE </td><td> 50mgの（4000 PU / MG分） </td></tr><tr><td>コラゲナーゼP </td><td> 85mg（1.78 U / mgのlyo） </td></tr><tr><td> SC2バッファ</td><td> 50ミリリットル</td></tr><tr><td colspan="2"> DNase I溶液 </td></tr><tr><td> DNase Iを</td><td> 6mgを（約2000U/mg） </td></tr><tr><td> GBSS - B </td><td> 3ミリリットル</td></tr><tr><td colspan="2"> ナイコデンツソリューション </td></tr><tr><td>ナイコデンツ</td><td> 8グラム</td></tr><tr><td> GBSS - </td><td> 28ミリリットル</td></tr></tbody></table> 表1。バッファと肝星細胞を分離するために必要な酵素のソリューション。すべてのバッファのpHは7.3から7.4に調整する必要があります。さらに、すべてのバッファの滅菌濾過することをお勧めします。特定の酵素活性に応じて使用される酵素の量を調整することが重要です。

<ol>
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肝星細胞は肝臓に不可欠な生理学的および病態生理学的プロセスを調節する。また、星状細胞は、それらを肝免疫応答の重要な構成要素をレンダリングする、抗原提示特性を有する。肝星細胞が肝臓の総細胞数の10〜15％を占めるが、これらの細胞の分離は、Disseの類洞周囲腔に局在のために挑戦している。 ここでは、 現場消化およびその後の勾配遠心分離の</e...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>商品番号</th></tr><tr><td> 70μmセルストレーナー</td><td> BD </td><td> 352350 </td></tr><tr><td>塩化カルシウム2 × 2H 2 O </td><td>シグマアルドリッチ</td><td> C3306 </td></tr><tr><td>コラゲナーゼP </td><td>ロッシュ</td><td> 11249002001 </td></tr><tr><td> DMEM </td><td>ギブコ</td><td> 11960 </td></tr><tr><td> DNase Iを</td><td>ロッシュ</td><td> 10104159001 </td></tr><tr><td> DPBS </td><td> Cellgro </td><td> 21から031 - CV </td></tr><tr><td> EGTA </td><td>フルカ</td><td> 3777 </td></tr><tr><td>ウシ胎仔血清</td><td>ギブコ</td><td> 16000 </td></tr><tr><td>グルコース</td><td>シグマアルドリッチ</td><td> G7528 </td></tr><tr><td>勾配遠心チューブ</td><td>グライナーバイオワン</td><td> 163160 </td></tr><tr><td> HEPES </td><td>ギブコ</td><td> 15630 </td></tr><tr><td> KCL </td><td>シグマアルドリッチ</td><td> P9541 </td></tr><tr><td> KH 2 PO 4 </td><td>シグマアルドリッチ</td><td> P9791 </td></tr><tr><td> L -グルタミン</td><td>ギブコ</td><td> 25030 </td></tr><tr><td>のMgCl 2 × 6H 2 O </td><td>フルカ</td><td> 63068 </td></tr><tr><td>し、MgSO 4 × 7H 2 O </td><td>シグマアルドリッチ</td><td> M2773 </td></tr><tr><td>のNa 2 HPO 4 × 2H 2 O </td><td>フルカ</td><td> 71643 </td></tr><tr><td> NaClの</td><td>シグマアルドリッチ</td><td> S3014 </td></tr><tr><td>のNaH 2 PO 4 × H 2 O </td><td>フルカ</td><td> 71507 </td></tr><tr><td> NaHCO 3の </td><td>フルカ</td><td> 71628 </td></tr><tr><td>ナイコデンツ</td><td> Accudenz AG </td><td> AN7050/BLK </td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td>ギブコ</td><td> 15140 </td></tr><tr><td>蠕動ポンプ</td><td>コー​​ルパーマー</td><td> HV - 7523〜70 </td></tr><tr><td>フェノールレッド</td><td>シグマアルドリッチ</td><td> P4633 </td></tr><tr><td>プロナーゼE </td><td>カルビオケム</td><td> 7433から2 </td></tr><tr><td>シリコンチューブ</td><td> Masterflex </td><td> HV - 96440〜14 </td></tr><tr><td>ピルビン酸ナトリウム</td><td>ギブコ</td><td> 11360 </td></tr><tr><td>組織培養フラスコ（25センチメートル2） </td><td> BD </td><td> 353108 </td></tr><tr><td>翼の輸液セット</td><td>テルモ</td><td> 1SV27EL </td></tr></tbody></table>

seven steps to stellate cells

肝星細胞は星状形態の肝臓常駐細胞であり、肝類洞内皮細胞と肝細胞の1,2の間にDisseの空間に配置されています。星状細胞は、骨髄の前駆体に由来し、全身のビタミン1、2の80％にまで保存されています。活性化されると、星状細胞は、このように肝線維症3に貢献し、細胞外マトリックスを生成するために筋線維芽細胞に分化する。契約する能力に基づいて、筋線維芽細胞星状細胞は、門脈圧亢進症4に関連血管緊張を調節することができる。最近、我々は、肝星細胞は、強力な抗原提示細胞であり、NKT細胞だけでなく、従来のTリンパ球5をアクティブにすることができます示した。 ここでは、マウスの肝臓から肝星細胞の効率的な製造方法を示す。そのperisinusoidalローカライズのために、肝星細胞の分離は、マルチステップのプロセスです。 Disseのスペースから分離へのアクセス星状細胞をレンダリングするためには、マウスの肝臓は、消化酵素のプロナーゼEとコラゲナーゼP.以下の血流とその場で灌流され、肝臓の組織は、プロナーゼEとコラゲナーゼP の持つ追加の酵素処理に供される試験管 。その後、このメソッドは、肝星細胞のビタミンA -を格納する脂肪滴の膨大な量を利用しています。この機能は8％ナイコデンツの勾配で遠心分離によって、他の肝細胞型から星状細胞を分離することができます。ここで説明するプロトコルは、星状細胞の非常に純粋で均一な人口をもたらします。調剤の純度は、蛍光顕微鏡またはフローサイトメトリーによる分析の前にマーカー分子グリア線維性酸性タンパク質（GFAP）、のために染色によって評価することができる。さらに、光学顕微鏡では脂肪滴を大量に抱いて星型肝星細胞のユニークな外観を明らかにする。 一緒になって、我々は、その形態学的外観および星状細胞の実体を定義するために役立つGFAPの発現の代表画像を含む肝星細胞の効率的な分離のための詳細なプロトコールを提示する。

Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

Seven Steps to Stellate Cells

ここでは、マウスの肝臓から肝星細胞の単離のための方法を説明します。星状細胞の精製のために、マウスの肝臓は、消化されています<em&gt;その場で</em&gt;と<em&gt; in vitroで</em&gt;密度勾配遠心分離の前にプロナーゼ、コラゲナーゼ処理による。この手法は、高純度の肝星細胞が得られます。

細胞星状に7つのステップ

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

Research

JoVE Journal

Immunology and Infection

33.5K ビュー.  Harvard Medical School. ここでは、マウスの肝臓から肝星細胞の単離のための方法を説明します。星状細胞の精製のために、マウスの肝臓は、消化されていますその場でと in vitroで密度勾配遠心分離の前にプロナーゼ、コラゲナーゼ処理による。この手法は、高純度の肝星細胞が得られます。

ビデオ: Seven Steps to Stellate Cells

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

全葉のイメージングを使用して細菌細胞をライブに気孔反応を評価する

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

免疫・感染学

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

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

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

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

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

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

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

水性を検出するタンジェンシャルフロー中空糸限外ろ過と熱解離のステップを使用し変更されたEPAメソッド1623<em&gt;クリプトスポリジウム</em&gt;と<em&gt;ジアルジア属。</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

ナイーブCD4のアデノウイルス形質導入Tregの分化を研究するためにT細胞

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

T細胞への抗原提示細胞による抗原の提示を研究するための抗原駆動性大腸炎モデルの開発

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

精密カットライブ肺樹状細胞を可視化するために、マウスの肺スライスを

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

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 ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

ヒトの過剰発現と精製<em&gt;シス</em&gt; - プレニルトランスフェラーゼ<em&gt;大腸菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

生体外でマウスのナチュラル キラー細胞の大量生産のため新規無料の送り装置システム

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

超高感度単一分子の開発と検証配列人間のインターフェロン-α のデジタルの酵素免疫測定法

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...