著者は彼女の技術支援のためのナンシーモーラに感謝したいと思います。 この作品は、ConsejoナシオナルデCiencia yをTecnologia、メキシコからの研究助成金48573-Mおよび168098でかつ助成IN212308とIN205311-2 Direccionから一般デAsuntosデルパーソナルAcademico、国立大学自治デ·メキシコ、メキシコによって賄われていた。

1。ヒトの血液から好中球の分離（PMN） <ol><li>抗凝固剤としてヘパリン（10 U / ml）で約20mlヒトの血液を使用します。血液はvenopunctureによって成人の健康なボランティアから採取した。 UNAM - 全ての実験は、のInstituto de InvestigacionesBiomédicasにおける生命倫理委員会の承認の下で行われた。 </li><li> 15mlコニカル遠心チューブにPBSで6％デキストランT500の2ミリリットルを入れて、血液の10ミリリットルを追加します。チューブを反転することによって2倍または3倍を混ぜて、それが赤血球沈降を可能にするために45分間放置します。 </li><li>新鮮な15mlコニカル遠心管に5ミリリットルのFicoll-Paqueは置く。 </li><li>赤血球沈降の上方に形成された白血球血小板血漿を取り、慎重に第2層を形成するのFicoll-Paqueの上に移します。 2フェーズがあることを確認してください。 </li><li> 4℃で20分間、516×gで遠心分離し、遠心分離後、血漿とのFicoll-Paqueの相間での単核細胞の層がある。ねーutrophilsは、チューブの下部にあります。 </li><li>上清を取り除き、ラックに対してチューブをタップして、細胞ペレットを壊し、風邪の10ミリリットル（4℃）、PBS（注）追加することで、細胞を再懸濁する。ピペッティングにより細胞を再懸濁し、これがあまりにもあるのでダウンはお勧めしませんが細胞にダメージを与える。 </li><li> 4℃で5分間、290×gで50ミリリットルコニカル遠心チューブと遠心分離機に細胞懸濁液を移す</li><li>前と同じように細胞ペレットを破ると冷たい（4℃）赤血球を溶解するために低張液（0.2％のNaCl、1％BSA、20mMのHEPES、pH = 7.4）中の10 mlを加える。正確に1分間手で優しくチューブを回して混ぜる。 </li><li>冷（4℃）高張液（1.6％のNaCl、1％BSA、20mMのHEPES、pH = 7.4）に、ミックス、血球計算盤（通常&gt; 95％はPMNである）を用いて細胞をカウント10mlを追加します。 注：細胞をカウントしながら氷上で細胞懸濁液をチューブに保管してください。 </li><li>以前と同じように遠心分離し、10℃PMNを再懸濁寒さの中7細胞/ ml（4℃）をPBS。氷上で保存する。 注：好中球が4で約6〜8時間、℃で生存可能で機能的なままである。 </li></ol> 2。 PMNのアクティブ化<ol><li> 1.5mlのエッペンドルフチューブにPMNの懸濁液100μl（1×10 7細胞/ ml）を入れてください。 注：サンプルチューブが少なくとも重複で行われるべきです。いつものネガティブコントロールのみのみ一次抗体、二次抗体を含むべきである。 </li><li> 10μg/ mlで対応する抗インテグリンまたは抗Fc受容体モノクローナル抗体（mAb）を追加し、氷上で15分間インキュベートする。 </li><li> 1 mlの冷（4℃）、PBS、遠心機で1743×gで（4,500 rpm）で遠心分離し、上清を吸引する。を追加することにより、PMNを洗う</li><li>ラックに対してチューブの底をタップすることで、細胞ペレットを壊し、1ミリリットルの冷PBSを追加し、2回以上前のステップのように洗ってください。 注：これらは結合していない抗体を除去洗う。 </li><li>同じで再懸濁し、PMN（内暖かいのitial）体積（37℃）Fの60μg/ mlの（ab &#39;）2ヤギ抗マウスIgGを含むPBS。 注：二次抗マウスIgG抗体は、最初の抗受容体抗体に結合し、受容体の架橋を引き起こすでしょう。 </li><li>興味のある核因子に応じて1〜20分、37℃でインキュベートする。 NF-κBは通常15分間、通常はエルク-1で5分間。 注：37℃で細胞をインキュベート4では行われません°C誘導する受容体架橋、℃ </li><li> 1ミリリットルの冷PBSを加え、遠心機で1743×gで3分間遠心する。 </li><li>上清を除去</li><li> dry-ice/ethanol浴ですぐにPMNを凍結し、10分間そこに保管してください。 </li></ol> 3。核の単離および固定<ol><li> 100μlの冷（4℃）低張緩衝液（10mMのHEPES、10mMのKCl、1.5mMのMgCl 2、再懸濁し、凍結PMNペレットと 1mMのたて付加DL-ジチオスレイトール[DTT];でpH = 7.9）。それが1つdry-ice/ethanol浴1からチューブを取り出して、そして低張緩衝液を加える前にチューブの底をきれいに拭くことをお勧めします。 </li><li>氷の上に置き、トリパンブルーによる核懸濁液のアリコートを染色することによって核の整合性をチェックします。核は円形と青に見える。無傷PMNは、ステンド取得しないと、細胞の破片は、不規則な形状の青色粒子として表示されます。 注：核懸濁液は、多くの無傷細胞やゴミが含まれている場合、それは準備を破棄し、別のサンプルを使用して手順を繰り返すことをお勧めします。 </li><li>寒い部屋の中で10分間遠心機で775×gで（3,000 rpm）で核を遠心分離します。非常に壊れやすく、余分であるこの時点で核ケアはサンプルは低温に保ち、過度の力で遠心分離していない場合は注意が必要です。 </li><li>非常に注意深くピペッティングにより上清を除去します。 </li><li>核を修正するために、PBS中の冷たい4％パラホルムアルデヒドを100μlを加える。 注：ペレットは非常に緩んでいるDバッファを追加することで核を再懸濁するのに十分です。上下にピペッティングとは避けるべきである。 </li><li>氷上で20分間インキュベートします。 </li><li> Immunolabelフローサイトメトリーのための核または4で最大24時間のためにそれらを保つ℃に</li></ol> 4。フローサイトメトリー分析のための核免疫標識<ol><li> 1分間遠心機で1743×gで（4,500 rpm）で核を遠心し、非常に穏やかにピペッティングにより上清を除去します。 </li><li>風邪を100μl（4℃）0.1％トリトンX-100、PBS中の4％パラホルムアルデヒド核を透過性にするために追加します。 10分間氷上でインキュベートする。 </li><li>遠心機で1743×gで透過処理した核を遠心分離し、上清を注意深く除去します。この時点で核ペレットがチューブの底にうまく接続しないでください。これは、ペレットが緩ん得れば再度遠心操作することをお勧めします。 </li><li>非特異的結合部位をブロックし、氷上で20分間インキュベートし、PBS中の冷4％ウシ胎児血清（FBS）500μlに再懸核。遠心遠心機で3分間1743 xgで核と慎重に上清を除去する。 </li><li> 4パーセントFBSおよび興味のある核因子に対するmAbの2.5μg/ mlを含有する冷100μlのPBSに再懸濁し核。氷上で20分間インキュベートします。 注：通常のネガティブコントロールのみ抗核因子抗体が含まれ、FITC標識二次抗体のみ必要があります。 </li><li> 4パーセントFBSおよび3分間1743 xgで遠心分離して冷PBS500μlで2回洗浄する。 </li><li> 、非常に慎重に4パーセントFBSおよび対応するFITC標識二次抗体10μg/ mlを含有する冷100μlのPBSに再懸濁した核を上清を除去し、氷上で20分間インキュベートします。 </li><li>前のステップのように4％FBSを冷PBS500μlで二回核を洗ってください。 </li><li> PBS中で冷4％パラホルムアルデヒド400μlの中の核を再懸濁します。 注：核は、サンプルが汚染することなく、数日間保存できるようにパラホルムアルデヒドに配置されている血清の存在下で発生する可能性のある問題。 </li><li> 4℃の暗所では、フローサイトメトリーやストアによって直ちに分析℃で3日間までのC。 </li></ol> 5。フローサイトメトリー分析<ol><li>または同様の装置、フローサイトメーターなどFACスキャン（フランクリンレイクス、ニュージャージー州Becton Dickinson社製）で免疫標識核を分析します。 </li><li> 196でログスケールでSSC、およびドットプロットのゲート核、10-1でログスケールでFSC：に取得設定を調整します。 </li><li>サンプルあたり1万核を獲得。 </li><li> 400℃で対数スケールに設定FL-1チャネル（506 nm）を介して、FITC-染色された核の蛍光分析。 </li></ol>

<ol>
	<li>Sendo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neutrophil+apoptosis:+its+biological+significance+in+inflammation+and+the+immune+response.">Regulation of neutrophil apoptosis: its biological significance in inflammation and the immune response.</a> Human Cell. 9, 215-222 (1996).</li><li>Borregaard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils,+from+marrow+to+microbes.">Neutrophils, from marrow to microbes.</a> Immunity. 33, 657-670 (2010).</li><li>Naussef, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+human+neutrophils+kill+and+degrade+microbes.+An+integrated+view.">How human neutrophils kill and degrade microbes. An integrated view.</a> Immunol. Rev. 219, 88-102 (2007).</li><li>Scapini, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neutrophil+as+a+cellular+source+of+chemokines.">The neutrophil as a cellular source of chemokines.</a> Immunol. Rev. 177, 195-203 (2000).</li><li>Hamilton, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-+and+stimulus-specific+mechanisms+for+post-transcriptional+control+of+neutrophil+chemokine+gene+expression.">Cell type- and stimulus-specific mechanisms for post-transcriptional control of neutrophil chemokine gene expression.</a> J. Leukoc. Biol. 91, 377-383 (2012).</li><li>Simon, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+apoptosis+pathways+and+their+modifications+in+inflammation.">Neutrophil apoptosis pathways and their modifications in inflammation.</a> Immunol. Rev. 193, 101-110 (2003).</li><li>Akgul, C., Moulding, D. A., Edwards, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+control+of+neutrophil+apoptosis.">Molecular control of neutrophil apoptosis.</a> FEBS Lett. 487, 318-322 (2001).</li><li>Witko-Sarsat, V., Pederzoli-Ribeil, M., Hirsch, E., Sozzani, S., Cassatella, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+neutrophil+apoptosis:+new+players+enter+the+game.">Regulating neutrophil apoptosis: new players enter the game.</a> Trends Immunol. 32, 117-124 (2011).</li><li>Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+and+NF-&#954;B+in+T+cell+activation:+life+at+the+edge.">Death and NF-&#954;B in T cell activation: life at the edge.</a> Mol. Cell. 11, 551-552 (2003).</li><li>Papa, S., Zazzeroni, F., Pham, C. G., Bubici, C., Franzoso, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+JNK+signaling+to+NF-&#954;B:+a+key+to+survival.">Linking JNK signaling to NF-&#954;B: a key to survival.</a> J. Cell Sci. 117, 5197-5208 (2004).</li><li>Valente, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF+increases+camptothecin-induced+apoptosis+by+inhibition+of+NF-&#954;B.">TNF increases camptothecin-induced apoptosis by inhibition of NF-&#954;B.</a> Eur. J. Cancer. 39, 1468-1477 (2003).</li><li>Choi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+NF-&#954;B+by+a+TAT-NEMO-binding+domain+peptide+accelerates+constitutive+apoptosis+and+abrogates+LPS-delayed+neutrophil+apoptosis.">Inhibition of NF-&#954;B by a TAT-NEMO-binding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis.</a> Blood. 102, 2259-2267 (2003).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+neutrophil+apoptosis+by+type+1+IFN+depends+on+cross-talk+between+phosphoinositol+3-kinase,+protein+kinase+C-d,+and+NF-&#954;B+signaling+pathways.">Inhibition of neutrophil apoptosis by type 1 IFN depends on cross-talk between phosphoinositol 3-kinase, protein kinase C-d, and NF-&#954;B signaling pathways.</a> J. Immunol. 171, 1035-1041 (2003).</li><li>Schnoor, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+non-viral+transfection+of+THP-1+cells.">Efficient non-viral transfection of THP-1 cells.</a> J. Immunol. Meth. 344, 109-115 (2009).</li><li>Garcia-Garcia, E., Rosales, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+factor+activation+by+Fc&#947;R+in+human+peripheral+blood+neutrophils+detected+by+a+novel+flow+cytometry-based+method.">Nuclear factor activation by Fc&#947;R in human peripheral blood neutrophils detected by a novel flow cytometry-based method.</a> J. Immunol. Meth. 320, 104-118 (2007).</li><li>Garcia-Garcia, E., Nieto-Castaneda, G., Ruiz-Saldana, M., Mora, N., Rosales, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc&#947;RIIA+and+Fc&#947;RIIIB+mediate+nuclear+factor+activation+through+separate+signaling+pathways+in+human+neuthophils.">Fc&#947;RIIA and Fc&#947;RIIIB mediate nuclear factor activation through separate signaling pathways in human neuthophils.</a> J. Immunol. 182, 4547-4556 (2009).</li></ol>

著者らは、彼らが競合する経済的利益を持っていないことを宣言します。

ここに記載の精製方法は、短時間で95％（顕微鏡観察により評価）、より高い純度を持つ非刺激好中球の分離（PMN）が可能になります。後者は完全に溶解されていない場合、時には好中球は、赤血球によって汚染することができます。赤血球及びPMNを簡単にフローサイトメトリーによって異なる細胞集団として区別することができるので、これは通常、技術に影響を与えません。隔離されたPM...

好中球は末梢血1に最も豊富に存在する白血球である。炎症や感染症、好中球の間に、彼らは防衛2の最初の行として働く場所患部に出現する最初の細胞である。好中球は貪食作用、活性酸素種の産生、脱顆粒によって溶菌酵素の放出、および4,5 -炎症性サイトカインの産生を含むいくつかの抗菌メカニズムの3を所有しています 。好中球は急速に様々な細胞表面の受容体からのシグナル伝達を介して活性化取得短命な細胞である。好中球は、その短い寿命のため、端末の細胞と考えられ、炎症過程6の間に活性化されない限り、彼らはアポトーシスを受けるので、それは、彼らはまた、特定の遺伝子の転写のレベルを変更することにより、それらの表現を変更できることが明らかになってきたが。サイトカイン5と7,8アポトーシスの阻害の生産は、i 2アール好中球の転写レベルで調節mportant活性化に依存した細胞機能。核因子κB（NF-κB）は、サイトカイン産生4の転写制御で、様々な種類の細胞における細胞生存とアポトーシス9-11の調節に関与している。 核因子の活性化につながるシグナル伝達経路は、通常、レポーター遺伝子アッセイにより、または電気泳動移動度シフトアッセイ（EMSA）によって研究されている。好中球は短命の細胞であるため、好中球のトランスフェクションのための効率的な技術が存在しないためしかし、これらの細胞における転写調節応答の研究は、レポーター遺伝子アッセイを使用して実行することはできません。 EMSAのアッセイは、核因子活性化12,13を探索するために好中球に使用されてきたが、それが放射性物質の使用を伴うので、しかしながら、この方法論は、複雑で高価である。クレオはsuccessfu使用されています別の技術であるLLY単球14をトランスフェクトする。したがって、少なくとも理論的には、核因子の活性化は、トランスフェクションによる好中球（低効率にもかかわらず）で検出することができた。しかし、この技術は、時間がかかり、おそらく少ない定量より高価になるだろう。免疫染色された細胞の顕微鏡分析はまた、核内で核内因子を検出するために使用することができる。実際、我々は核 ​​こうして15にNF-κBの転座を検出した。残念なことに、この手法はまた、時間のかかるレス定量的、かつ観察者バイアスの影響を受けます。 ここでは、フローサイトメトリーによって単離し、免疫標識核における核内因子の検出および定量化を可能にするシンプルで効率的な方法を提示する。我々は、F（フローサイトメトリーにより、ヒト末梢血から好中球を分離抗受容体抗体とインテグリンまたはFc受容体を介して、これらの細胞を刺激し、隔離し、immunolabel核を、核を分析するための手法について説明igure 1）。メソッドが成功し、NF-κB15および好中球の核内エルク-1 16核内因子を検出するために使用されています。この方法の感度は、核内の核因子レベルの小さな変化を検出することができます。また、この方法は、他の細胞型から核内転写因子のレベルを分析するために使用することができます。

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td >Heparin </td>
 <td >PiSA (Mexico)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td >Dextran T500</td>
 <td >Pharmacosmos A/S (Holbaek, Denmark) </td>
 <td>T1-Dextran T500</td>
 <td></td>
 </tr>
 <tr>
 <td >Ficoll-Paque</td>
 <td >Pharmacia</td>
 <td>17-0320-01 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium chloride </td>
 <td >Sigma</td>
 <td>S7653 </td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate monobasic</td>
 <td >Sigma</td>
 <td>S9638</td>
 <td></td>
 </tr>
 <tr>
 <td >Sodium phosphate dibasic</td>
 <td >Sigma</td>
 <td>S9390</td>
 <td></td>
 </tr>
 <tr>
 <td >Bovine serum albumin (BSA)</td>
 <td >Sigma</td>
 <td>A2153</td>
 <td>Cohn Fraction V</td>
 </tr>
 <tr>
 <td >HEPES</td>
 <td >Sigma</td>
 <td>H3375 </td>
 <td></td>
 </tr>
 <tr>
 <td >Potassium chloride</td>
 <td >Sigma</td>
 <td>P9541 </td>
 <td></td>
 </tr>
 <tr>
 <td >Magnesium chloride anhydrous </td>
 <td >Sigma</td>
 <td>M8266 </td>
 <td></td>
 </tr>
 <tr>
 <td >DL-dithiothreitol (DTT) </td>
 <td >Sigma</td>
 <td>D9163</td>
 <td></td>
 </tr>
 <tr>
 <td >Trypan Blue (0.4 % solution)</td>
 <td >Sigma</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td >Paraformaldehyde</td>
 <td >Sigma</td>
 <td>P6148 </td>
 <td></td>
 </tr>
 <tr>
 <td >Triton X-100 </td>
 <td >Sigma</td>
 <td>X100</td>
 <td></td>
 </tr>
 <tr>
 <td >Fetal bovine serum (FBS)</td>
 <td >GIBCO</td>
 <td>10437-028</td>
 <td></td>
 </tr>
 <tr>
 <td >Monoclonal antibody IV.3</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>025-1 </td>
 <td>Human-specific anti-FcRII (CD32) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody 3G8</td>
 <td >Medarex (Annandale, NJ) </td>
 <td>028-2 </td>
 <td>Human-specific anti-FcRIII (CD16) </td>
 </tr>
 <tr>
 <td >Monoclonal antibody TS2/16</td>
 <td >Dana Farber Cancer Research Institute (Boston, MA) </td>
 <td>Donated by Dr. Martin Hemler </td>
 <td>Human-specific anti-&beta;1 integrin (CD29)</td>
 </tr>
 <tr>
 <td >Monoclonal antibody IB4</td>
 <td >University of California, San Francisco </td>
 <td>Donated by Dr. Eric J. Brown </td>
 <td>Human-specific anti-&beta;2 integrin (CD18)</td>
 </tr>
 <tr>
 <td >F(ab')2 goat anti-mouse IgG </td>
 <td >Cappel (Aurora, OH)</td>
 <td>55468</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-mouse IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55522</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC-conjugated F(ab')2 goat anti-rabbit IgG</td>
 <td >Cappel (Aurora, OH)</td>
 <td>55665</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p50</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-114</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td>Anti-NF-&kappa;B p65</td>
 <td >Santa Cruz Biotechnology (Santa Cruz, CA)</td>
 <td>sc-109</td>
 <td>Rabbit polyclonal antibody</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>15-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430791</td>
 <td></td>
 </tr>
 <tr>
 <td>50-ml centrifuge tube</td>
 <td >Corning</td>
 <td>430291</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge, Sorvall Tabletop</td>
 <td >Dupont Instruments</td>
 <td>RT 6000D</td>
 <td></td>
 </tr>
 <tr>
 <td>pH-meter</td>
 <td >Corning</td>
 <td>340</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-20</td>
 <td >Gilson </td>
 <td>F123600</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-200</td>
 <td >Gilson </td>
 <td>F123601</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetman pipette P-1000</td>
 <td >Gilson </td>
 <td>F123602</td>
 <td></td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td >Fisher Scientific</td>
 <td>0267110</td>
 <td></td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td >Nikon </td>
 <td>Eclipse E600</td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope</td>
 <td >Nikon </td>
 <td>TMS</td>
 <td></td>
 </tr>
 <tr>
 <td>Water Bath Incubator</td>
 <td >Fisher Scientific </td>
 <td>2IS-M</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5414C</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td >Eppendorf</td>
 <td>5418</td>
 <td></td>
 </tr>
 <tr>
 <td>Flow Cytometer</td>
 <td >Becton Dickinson (Franklin Lakes, NJ) </td>
 <td>FACScalibur </td>
 <td></td>
 </tr>
 </tbody>
</table>

a simple and efficient method to detect nuclear factor activation in human neutrophils by flow cytometry

好中球は、末梢血中に最も豊富に存在する白血球である。これらの細胞は、このように微生物を侵略に対する最初の防衛線となって、炎症や感染部位に最初に表示されています。好中球は、貪食作用、溶菌酵素の放出、活性酸素種の産生などの重要な抗菌機能を有する。これらの重要な防御機能に加えて、好中球などの炎症性サイトカインおよびアポトーシスの阻害の生産などの感染症に対応して他のタスクを実行します。サイトカインは、感染をクリアに役立つ他の白血球をリクルートする、アポトーシスの阻害は、好中球が感染部位で長生きすることができます。これらの機能は、転写レベルで調節されている。好中球は短命の細胞であるため、全く効率が存在しないためしかし、これらの細胞における転写調節応答の研究は、従来のレポーター遺伝子のメソッドで実行することはできません好中球のトランスフェクションのためcientテクニック。ここでは、フローサイトメトリーによって単離し、免疫標識核における核内因子の検出および定量化を可能にするシンプルで効率的な方法を提示する。我々は、ヒト末梢血から純粋な好中球を分離するための手法について説明抗受容体抗体で、これらの細胞を刺激し、隔離し、immunolabel核、フローサイトメトリーで核を分析します。メソッドが成功した好中球および他の細胞型から核内にNF-κBおよびエルク-1核内因子を検出するために使用されています。したがって、この方法では、種々の細胞型から単離核内転写因子の活性を分析するためのオプションを表します。

ここに記載の精製方法は、通常は95％（ 図1A）を超える純度を有する非刺激好中球（PMN）を提供しています。隔離されたPMNはその後、特定のモノクローナル抗体で架橋する特定の受容体によって刺激することができる。我々は、Fc受容体とインテグリン（ 図1B）を介してPMNを刺激してきた。一度刺激、PMNを溶解し、核を高収率で分離されています。核は、そのような?...

Watch this Scientific Journal Video about A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry at JoVE.com

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

好中球は、血液中に最も豊富に存在する白血球である。好中球は、このような炎症性サイトカインとアポトーシスの抑制​​の生産などの転写調節機能を有する。これらの関数は、単離核で、フローサイトメトリーによって核内因子の検出および定量化を可能にするここに示す方法で研究することができる

フローサイトメトリーによるヒト好中球の核因子の活性を検出するために簡単で効率的な方法

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

a-simple-efficient-method-to-detect-nuclear-factor-activation-human

Research

JoVE Journal

Immunology and Infection

17.7K ビュー.  University of Alberta. 好中球は、血液中に最も豊富に存在する白血球である。好中球は、このような炎症性サイトカインとアポトーシスの抑制​​の生産などの転写調節機能を有する。これらの関数は、単離核で、フローサイトメトリーによって核内因子の検出および定量化を可能にするここに示す方法で研究することができる

ビデオ: A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

フローサイトメトリーによって負傷した脊髄組織における免疫細胞の定量的評価：セルの精製方法のための新しい使用法

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

免疫・感染学

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

成人肝細胞の混合初代培養からマクロファージを分離するためにシンプルかつ効率的な方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

ヒトのインビトロ</em免疫バランスの初期状態の認識のためのスクリーニングツールとして>抑制

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

フローサイトメトリーによる胸腺正と負の選択の検討

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

の相互作用を研究<em&gt;黄色ブドウ球菌</em&gt;フローサイトメトリーとタイムラプス顕微鏡による好中球と

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

簡単かつ迅速なプロトコル非酵素的に浸潤リンパ球の分析のための新鮮なヒト組織を解離

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

突然変異体ワクシニアウイルスベクターを構築するためのシンプルで効率的なアプローチ

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

に応答したヒト一次好中球及び単球による抗カビ活動のライブイメージング<em&gt; A。フミガーツス</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

離とマウスとヒトの肺胞マクロファージの体外培養

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血によって人間の Monocyte のサブセットの解析フローのフローサイトメトリー解析

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...