Name: neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents
Uploaded: 2016-03-25T15:00:00.000+00:00
Duration: 14 min 4 s
Description: Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

This work was supported by the Institute National du Cancer (INCa-DGOS-4664).

初代白血病細胞1.好中球の単離と共培養注：手順は、すべての患者がインフォームドコンセントに署名リヨン病院倫理委員会の承認の下で行いました。 <ol><li> 初代白血病細胞と好中球の単離 <ol><li>慢性リンパ性白血病（CLL）と診断された患者からEDTA（血液1ミリリットル当たり1.8ミリグラムのEDTA）に末梢血のチューブを収集します。 </li><li>滅菌50mlチューブに血液の各15ミリリットルを追加し、15ミリリットルRPMI（希釈1：1）で希釈し、その後、慎重にゆっくりと相を混合することなく、チューブの底に密度勾配溶液15mlを加えます。密度勾配液を調製するためにRTであることを確認してください。 </li><li> RTでブレーキなしで35分間300×gで遠心分離します。血液は、 図1（a）に示すように 、4つの別個の相に分離底部へ戻るべきである：血小板および血漿、単核細胞（Wハイト環）、密度勾配溶液、顆粒球および赤血球。 </li><li>プラスチック製パスツールピペットと新しい50mlチューブへの転送と、一次白血病細胞を表す白いリングを収集します。 <ol><li> RTで10分間、300gで合計し、遠心分離機で50ミリリットルまでのPBS（カルシウムおよびマグネシウムを含有する）でチューブを埋めます。 </li><li> PBSは、その後、室温で10分間、300グラムのPBSを合計し、遠心分離機で最大50ミリリットルを追加5mlでペレットを再懸濁。 </li><li>完全RPMI培地でペレットを再懸濁（RPMI 1640、10％ウシ胎児血清（FBS）、2mMグルタミン、100 Uを補充/ mlのペニシリンおよび100mg / mlストレプトマイシン）細胞生存率カウンターを用いて計数します。 </li></ol></li><li>その後、合計で50ミリリットルに0.9％NaClupの3％デキストランを追加顆粒球および赤血球相を残して、上部相を吸引し、25ミリリットルまでのPBSを追加します。チューブを10回混合し、混合することなく、室温で30分間保管してください。 </li><li>上位RBC-乏しい好中球層ANを収集清潔な無菌50mlチューブ中に置き、室温で10分間、300gで管を遠心dは。 PBSは、その後、合計50 mlまでの赤血球溶解緩衝液を追加5mlで再懸濁し、各ペレット。 </li><li> RTで10分間、500gで遠心分離後、室温で15分間、暗所でチューブを保管してください。次いで、室温で10分間、500gで遠心分離する（合計で50ミリリットルまでのPBSを追加）PBSでペレットを洗ってください。 </li><li>細胞生存率カウンターを用いてカウントするための完全RPMI培地でペレットを再懸濁します。フローサイトメトリーを用いて、それらの単離の純度を決定するために、5mlの50μlのPBS-FBS（4％）にプラスチックチューブを、各細胞型の3×10 5を保ちます。 </li></ol></li><li> 単離された細胞集団の形態学的外観を分析 <ol><li>これを行うには、それぞれ3×10 4の好中球および150μlの完全RPMI培地中で3×10 4の単核細胞を再懸濁します。そのTを確実に、サイトスピン遠心分離機ガラススライド、フィルタカード、およびサンプルチャンバを組み立てます彼はよくバランスが取れて遠心します。 </li><li> RTで10分間、750×gで試料室とCYTO-遠心機に各細胞型を置き、遠心分離機からスライドし、フィルタカード、およびサンプル室を削除します。スライド上の細胞を損傷しないように、慎重に分解します。フィルタカードとサンプル室を捨てます。 </li><li>ギムザ染色キットを用いて細胞を染色し、乾燥さ1時間のスライドを保ちます。 100Xの倍率の顕微鏡で細胞を調べます。 </li></ol></li><li> FACSによって単離された細胞の純度をテスト <ol><li> APCに結 ​​合させた抗ヒトCD19抗体（5μL/ 10 6細胞）を用いて、単離された一次白血病細胞にラベルを付けます。 表1に記載されている抗ヒト抗体（5μL/ 10 6細胞）の混合物で精製した好中球にラベルを付けます。 4 O℃で30分間、暗所で細胞をインキュベート</li><li> 500μlのPBS-FBS（4％）、室温で5分間300グラムでチューブを遠心分離で細胞を洗浄。 </li><li>200μlのPBS-FBS（4％）の各ペレットを再懸濁。 </li><li> 、SSC-A（488 / 10BP）、FITC（BP 530/30）、PerCPを-のCy5.5（40分の695 BP FSC-A（488 nM）を：488 nmレーザー：以下の光学構成を使用して、フローサイトメーターで分析）、PE（BPを575/26）; 633 nmレーザー：APC（BPを660/20）、APC-Cy7の（BPを780/60）。色補正を確認してください。 </li></ol></li><li> 自家好中球との一次白血病細胞の共培養 <ol><li>シード一次白血病細胞の2×10 5細胞/ ml単独または完全RPMI培地中で比1:10での自己好中球です。これを行うには、細胞懸濁液を希釈して細胞数を調整し、2×10 6細胞/ mlの好中球への一次白血病細胞の2×10 5細胞/ mlを追加します。慎重に混ぜます。 </li><li>細胞にブルートンチロシンキナーゼ（Btk）阻害剤（ibrutinib）の25μMを追加します。 5％CO 2、37℃で24時間インキュベートします。 </li></ol></li><li> セルハーベストおよびFACS分析 <ol><li>コルECT FACS分析5mlのプラスチックチューブ中の細胞、室温で5分間、300gで管を遠心。 1mlのPBS-FBSでペレットを洗浄し（4％）、次いで、室温で5分間300gで遠心分離します。 </li><li>市販のキットを用いて、アネキシンVおよびPIでの再懸濁ペレットとRTで10分間、暗所で細胞をインキュベートします。 488nmレーザー：FSC-A（488 nM）を、SSC-A（488 / 10BP）、FITC（BPを530/30）、PI（610、図2Aおよび以下の光学構成のゲート戦略を使用して、フローサイトメーターで分析/ 20 BP）。色補正を確認してください。 </li></ol></li></ol>顆粒球経路と3DモデルでRLリンパ腫B細胞との共培養に沿ってHL60細胞の2分化<ol><li> 好中球様細胞にヒト前骨髄球性（HL60）細胞の分化 <ol><li> 3×10 5細胞/ mlにHL60細胞数を調整し、その後、それらの分化を誘導するために、1μMのレチノイン酸（RA）および1.25％DMSOを加えます。各3×10 5をシード</s48ウェルプレート中のウェルあたりHL60細胞は、5％CO 2、37℃で48時間インキュベート&gt;まで。 </li><li>その後、室温で5分間300グラムで細胞と遠心分離を収集します。細胞生存率カウンターを用いて計数する完全RPMI培地で細胞を再懸濁します。 </li><li>次いで、3×10 5細胞/ mlにHL60細胞数を調整し、1μMのレチノイン酸（RA）および1.25％DMSOを添加することによって再び分化を誘導するために、完全RPMI培地で細胞懸濁液を希釈します。ウェルあたり48ウェルプレートの各3×10 5個のHL60細胞を播種し、5％CO 2、37℃でさらに48時間インキュベートします。 </li></ol></li><li> HL60分化の解析（HL60 デフ ） <ol><li>細胞を収集し、室温で5分間300グラムでチューブを遠心します。細胞生存率カウンターを用いて計数する完全RPMI培地でペレットを再懸濁。 </li><li>フローサイトメトリーによる細胞表面マーカー発現の変化を分析します。各3×10 5を配置</suFACS分析のため（細胞培養物から）、P&gt;未分化のHL60細胞および5ml中3×10 5 HL60 差分細胞プラスチックチューブ、室温で5分間、300gで管を遠心。 </li><li> 50μlのPBS-FBS（4％）でペレットを再懸濁します。 AF700またはAPCに結 ​​合させた抗ヒトCD38（5μL/ 10 6細胞）に結合した抗ヒトのCD11bと細胞を標識。 4 O℃で30分間、暗所で細胞をインキュベート</li><li>その後、室温で5分間、300gで遠心分離（4％）、500μlのPBS-FBSで洗浄します。 200μlのPBS-FBS（4％）でペレットを再懸濁します。 </li><li> 488nmレーザー： 図3Aおよび以下の光学構成のゲート戦略を使用して、フローサイトメーター上で分析FSC-A（488 nM）を、SSC-A（488 / 10BP）。 633 nmレーザー：APC（660/20 BP）、アレクサフルオロ700（BPを730/45）。 </li><li> HL60 デフの形態変化をテストするには、顕微鏡検査のためのスライドガラス上に細胞を固定化します。 <ol><li>これを行うには、それぞれ3×10を再懸濁<su（細胞培養物から）のp&gt; 4未分化のHL60細胞および150μlの完全RPMI培地中で3×10 4 HL60 デフ 。遠心分離機はバランスが取れていることを確認して、サイ​​トスピン遠心分離機ガラススライド、フィルタカード、およびサンプルチャンバを組み立てます。 </li><li> RTで10分間、1,500rpmで試料室とCYTO-遠心機に各細胞型を置きます。遠心分離機からのスライド、フィルタカード、およびサンプル室を削除します。スライド上の細胞を損傷しないように、慎重に分解します。フィルタカードとサンプル室を捨てます。 </li><li>ギムザ染色キットを用いて細胞を染色し、乾燥さ1時間のスライドを保ちます。 100Xの倍率の顕微鏡で細胞を調べます。 </li></ol></li></ol></li><li> 3次元（3D）培養 注：この実験で用いた材料が冷えていることを確認し、実験が氷の上で行われています。 <ol><li>単独で、またはHL60 デフと混合のいずれかで、それぞれ5×10 4 RL細胞を再懸濁</em&gt;比RLで：HL60 デフ午前1時10分、2〜3mm程度に開口部を広げるために、滅菌はさみで切断し1ミリリットルピペットチップを使用して、300μlの基底膜マトリックスを持ちます。このステップの間に気泡を避けてください。 </li><li> 24ウェルプレート中の細胞懸濁液/各300μLをシード。このステップの間に気泡を避けてください。次いで、各ウェルについてRPMI培地を完了し、5％CO 2、37℃で7日間インキュベートML 1を追加、5％CO 2で37℃で30分間、プレートをインキュベートします。 2日毎に培地を変更します。 5日目に10 nMのビンクリスチンを追加します。 </li></ol></li><li> FACS分析 <ol><li> 7日間の培養後、培地を吸引し、二回1ミリリットルの氷冷PBSで各ウェルを洗浄します。氷冷PBS-EDTA（5ミリモル）の3ミリリットル/ウェルを追加します。 200μlのピペットチップの底を使用して、こすることによってウェルの底からゲルを外します。氷上で30分間静かにプレートを振ります。 </li><li>滅菌15ミリリットルチューブに細胞懸濁液を移し、ICを軽くチューブを振ります別の30分間の電子。均一な細胞懸濁液の外観を確認してください。 （それはケースではない場合には、長い時間のための細胞を振る以上のPBS-EDTAを追加します）。 </li><li> RTで10分間、300×gでチューブを遠心。その後、室温で10分間、300×gで遠心分離し、PBSでペレットを洗ってください。 </li><li> PBS-FBS（4％）で再懸濁し、PE-Cy7をし、APCに結 ​​合した抗ヒトCD38抗体（5μL/ 10 6細胞）を結合させた抗ヒトCD19抗体で標識します。 4 O℃で30分間、暗所でインキュベート</li><li> RTで5分間300gで、次いで遠心チューブ500μlのPBS-FBS（4％）で洗浄しました。市販のキットを使用してアネキシンVおよびPIで細胞を再懸濁します。 </li><li> 図4Aのゲート戦略を使用して、フローサイトメーター上で分析し、以下の光学構成：488 nmレーザー：FSC-A（488 nM）を、SSC-A（488 / 10BP）、FITC（530/30 BP）、PI（610 / 20 BP）、PE-Cy7の（780/60 BP）。 633 nmレーザー：APC（BPを660/20）。色補正を確認してください。 </li></ol></li&gt; 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessories+to+the+crime:+functions+of+cells+recruited+to+the+tumor+microenvironment.">Accessories to the crime: functions of cells recruited to the tumor microenvironment.</a> Cancer Cell. 3, 309-322 (2012).</li><li>Shojaei, F., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refractoriness+to+antivascular+endothelial+growth+factor+treatment:+role+of+myeloid+cells.">Refractoriness to antivascular endothelial growth factor treatment: role of myeloid cells.</a> Cancer Res. 68, 5501-5504 (2008).</li><li>Liang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+promote+the+malignant+glioma+phenotype+through+S100A4.">Neutrophils promote the malignant glioma phenotype through S100A4.</a> Clin Cancer Res Off J Am Assoc Cancer Res. 20, 187-198 (2014).</li><li>Teramukai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+neutrophil+count+as+an+independent+prognostic+factor+in+advanced+non-small-cell+lung+cancer:+an+analysis+of+Japan+Multinational+Trial+Organisation+LC00-03.">Pretreatment neutrophil count as an independent prognostic factor in advanced non-small-cell lung cancer: an analysis of Japan Multinational Trial Organisation LC00-03.</a> Eur. J. Cancer Oxf.Engl 1990. 45, 1950-1958 (2009).</li><li>Zhu, Q., 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+IL-6-STAT3+axis+mediates+a+reciprocal+crosstalk+between+cancer-derived+mesenchymal+stem+cells+and+neutrophils+to+synergistically+prompt+gastric+cancer+progression.">The IL-6-STAT3 axis mediates a reciprocal crosstalk between cancer-derived mesenchymal stem cells and neutrophils to synergistically prompt gastric cancer progression.</a> Cell Death Dis. 5, 1295 (2014).</li></ol>

The authors have nothing to disclose.

また、分離回収され、密度勾配遠心分離法を用いて高純度のヒト血液から好中球の単離のために、同じ手順単核細胞内で、ここに有効な、簡単、迅速かつ安価なプロトコルを記載しています。単離された細胞集団は、≥90％純粋です。 いくつかの方法がヒトの血液から好中球の単離のために用意されています。これらは、不連続勾配11,12を使用して、または好中?...

自然免疫細胞は、腫瘍微小環境内の細胞の本質的な割合を構成し、患者や癌1の動物モデルにおいて腫瘍の悪性度と関連しています。最近では、より広く、慢性免疫応答は、化学療法2の腫瘍の進行、転移および抵抗の促進に重要な役割を果たしていることは理解となっています。マクロファージは、直接化学療法3,4に対する腫瘍細胞の応答を調節することが示されてきた重要な自然免疫細胞です。しかしながら、抗癌治療に対する腫瘍応答の調節に好中球、先天性免疫系において重要なプレーヤーの役割は、不明です。これらのプロトコルの目的は、CLL患者の血液サンプルからの好中球を分離し、抗リンパ腫剤に対するリンパ腫細胞の感受性の調節におけるそれらの役割を研究するために、顆粒球経路に沿ったHL60細胞を区別するために、高速かつ信頼できる方法を使用することです。 6の侵入に対する防御の第一線としての血液5と行為における自然免疫系の最も豊富な細胞成分です。好中球は、いくつかの病的状態7に可変エフェクター機能に加えて、効果的な自然免疫応答を上昇中で重要な役割を持っています。したがって、このような密度勾配分離法などの他の血液細胞から好中球を単離するための迅速かつ信頼できる方法は、in vitro試験のために必要とされます。好中球の単離のために、このメソッドを使用すると、インビボおよびex vivo での好中球媒介性の免疫機能に関するさらなる研究を促進します。 好中球の純粋な集団を得る能力は、免疫学的疾患8を有する患者の調査のための重要な第一歩です。密度勾配分離法は、細胞の高い収率が得られる理想的な技術です。メソジスト教徒dは休憩なしで35分間300gで遠心分離した希釈したヒト血液を含む管の底に密度勾配溶液を添加することを含みます。単核細胞のリングは、インターフェイスに表示され、好中球は、前者の下に常駐します。この方法は、はるかに9高価である好中球単離キットなど、他の利用可能な方法に対して大きな利点を持っています。また、ヒト好中球に特異的な表面マーカーに対する抗体を用いて市販のキットによって、ヒト血液から好中球を単離し、細胞の活性化または分化のリスクを高めます。密度勾配分離法は、短時間での好中球の単離を可能にします。同じステップ内では、単核細胞はまた、分離回収されています。これは、純粋な細胞の高い収率を機能的完全性を実現するために取得された基本的な技術です。 腫瘍microenvを模倣するために、ironment、3D実験を行いました。 in vitroでの好中球の半減期が短い考えると、新鮮なヒト好中球と3D実験は決定的なものではありません。このため、前骨髄球（HL60）細胞を、分化誘導剤、ジメチルスルホキシド（DMSO）及びレチノイン酸（RA）を使用して好中球様細胞に分化するように誘導されます。分化したHL60細胞（HL60 デフ）を使用すると、異なるドナーからの単離に起因する好中球の異なる応答を持つ防ぐことができます。 試験管 3D で培養モデルは、in vitroでの2Dモデル間およびin vivoモデル中間段階を表します。 2D培養では、細胞は、この合成表面に変性された堆積タンパク質への不自然な細胞の添付ファイルを形成するプラスチック表面に広がります。逆に、細胞を、それらが合成細胞外マトリックスからの3D培養フォーム天然細胞間添付の細胞は、それらが結合している天然物質です。この理由のために、特に癌細胞および他の細胞型との間の三次元共培養モデルは、腫瘍増殖、血管形成、および転移への寄与を示すために非常に有用でした。その結果、三次元培養は、細胞培養は、 インビボ 10 に存在する生理学的条件を模倣します。

<table><tbody><tr><td>RPMI 1640&amp;nbsp;</td><td>Gibco Invitrogen</td><td>21875-034</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco Invitrogen</td><td>10270-106</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS contains calcium and magnesium)</td><td>Gibco Invitrogen</td><td>14040-091</td><td></td></tr><tr><td>N-acetyl-L-alanyl-L-glutamine (L-Glutamine)</td><td>Life technologies</td><td>25030-024</td><td></td></tr><tr><td>Penicillin streptomycin (Pen Strep)</td><td>Life technologies</td><td>15140-122</td><td></td></tr><tr><td>Bruton&#039;s tyrosine kinase (Btk) inhibitor (Ibrutinib)&amp;nbsp;</td><td>CliniSciences</td><td>A3001</td><td></td></tr><tr><td>Vincristine&amp;nbsp;</td><td>EG labo</td><td></td><td></td></tr><tr><td>BD Matrigel basement membrane matrix&amp;nbsp;</td><td>BD Biosciences</td><td>354234</td><td>Put at 4 &lt;sup&gt;o&lt;/sup&gt;C overnight (O/N) before the day of the experiment</td></tr><tr><td>Red cell lysis buffer&amp;nbsp;</td><td>BD Biosciences</td><td>555899</td><td></td></tr><tr><td>Ficoll (Pancoll)</td><td>PAN Biotech</td><td>P04-60500</td><td></td></tr><tr><td>Dextran&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8906</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma-Aldrich</td><td>R2625</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Euromedex</td><td>S3014</td><td></td></tr><tr><td>Annexing V-FLOUS staining kit&amp;nbsp;</td><td>Roche</td><td>11&amp;nbsp;988&amp;nbsp;549&amp;nbsp;001</td><td></td></tr><tr><td>Kit RAL 555 Modified Giemsa staining kit</td><td>Cosmos Biomedical</td><td>CB361550-0000</td><td></td></tr><tr><td>LSRII flow cytometry</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Cytocentrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Leica DMR-XA microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Cellometer Auto T4 Cell Viability Counter</td><td>Nexcelom&amp;nbsp; Bioscience</td><td></td><td></td></tr></tbody></table>

neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

白いリングは、単核細胞を表し、血小板及び血漿：ここに記載の密度勾配分離法は、CLL患者の血液から単離した一次白血病細胞と非刺激好中球は、 図1Aは、上から下に（密度勾配遠心分離後に得られた異なる血液層を表し、提供します密度勾配溶液、顆粒球および赤血球）。 図1B及び1Cは、それぞれ好中球（マルチローブ核細胞）と単核細胞?...

Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

neutrophil-isolation-analysis-to-determine-their-role-lymphoma-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

15.2K ビュー.  Cancer Research Center in Lyon. Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents. 

ビデオ: Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

低密度の好中球によって生成された好中球細胞外トラップは仲介する腹腔洗浄液から得られた腫瘍細胞の増殖と添付ファイル

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

がん研究

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

慢性リンパ性白血病の新規 NFAT2 ターゲット遺伝子を識別するクロマチン免疫沈降法

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

主な白血病細胞の代謝プロファイルの評価

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

HPLCベースのアッセイは、ヒト慢性リンパ球性白血病細胞における細胞外ヌクレオチド/ヌクレオシド代謝を監視します

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

化学

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

核/細胞質タンパク質の密売を監視する原発性慢性リンパ球性白血病細胞の細胞分画

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

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 ...

免疫・感染学

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

抗腫瘍特性を有する好中球の単離とキャラクタリゼーション

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

タンパク質機能に2DE  - ゲルスポットから：慢性リンパ性白血病で、HS1から学んだレッスン

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

医学

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

リンパ球における解糖およびミトコンドリア呼吸を分析するために最適化されたプロトコル

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

The Isolation and Characterization of Low- and Normal- Density Neutrophils from Whole Blood

Emerging research shows that the circulating neutrophil population in humans consists of diverse subtypes and should not be studied as a single population, as has been done historically. In particular, low-density and normal-density neutrophils (LDNs, NDNs) have been shown to have functionally and metabolically distinct profiles, a factor that must be considered when publishing neutrophil research. Here, we present a modified method for the untouched isolation and separation of LDNs and NDNs from whole blood.
The density gradient medium (1.135 g/mL) is combined at 9:10 with 10x PBS. Specific density gradients of 55%, 70%, and 81% are subsequently made by combining the 100% density gradient medium with 1x phosphate-buffered saline (PBS). Neutrophils isolated from 12 mL of peripheral whole blood obtained from consented donors using a negative selection-based magnetic isolation kit are resuspended in the 55% fraction. A volume of 3 mL of the 81% and 70% fractions is layered into a 15 mL tube, followed by the 55% fraction containing total neutrophils. The density gradients are then centrifuged at 720 x&#160;g for 30 min. Two distinct bands are obtained at the 55%/70% interface (LDNs) and 70%/81% interface (NDNs). The cells are carefully pipetted into separate tubes and washed using PBS. The purity of the isolated fractions is determined using flow cytometry. Both LDNs and NDNs were defined as CD14lo CD15+ SSChi by flow cytometry. Isolation purity was calculated at &#8805;93% of viable cells for both types.
This method provides a reliable and efficient approach for separating LDN and NDNs from peripheral blood, ensuring high purity and viability of the isolated cells. Enhancing the precision of neutrophil isolation facilitates more accurate downstream analyses of these distinct neutrophil subpopulations. These are critical for advancing our understanding of neutrophil heterogeneity and its implications in various physiological and pathological contexts.

Here, we provide a reliable approach for isolating low- and normal-density neutrophils from whole blood using magnetic isolation (negative selection) and discontinuous density gradient medium. It ensures untouched isolation of high-purity cells (&#8805;93%), facilitating accurate downstream analysis of neutrophil subpopulations, crucial for understanding their roles in health and disease.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

要約

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