ZGはイスラエル科学財団（助成金番号11分の41）のI-COREプログラムからの助成金によってサポートされ、Abisch·フレンケル財団、Rosetrees信託、イスラエル癌研究財団（ICRF - 研究キャリア開発賞）とCONCERNファンデーション。 ZGFはイスラエル癌研究財団（ICRF - 研究キャリア開発賞）からの助成金によってサポートされ、保健のイスラエル省イスラエル肺協会のチーフサイエンティスト。

<ol>
	<li>Tangye, S. G., Brink, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+helping+hand+from+neutrophils+in+T+cell-independent+antibody+responses.">A helping hand from neutrophils in T cell-independent antibody responses.</a> Nat Immunol. 13, 111-113 (2012).</li><li>Mantovani, A., Cassatella, M. A., Costantini, C., Jaillon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+in+the+activation+and+regulation+of+innate+and+adaptive+immunity.">Neutrophils in the activation and regulation of innate and adaptive immunity.</a> Nat Rev Immunol. 11, 519-531 (2011).</li><li>Pruijt, J. 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=Neutrophils+are+indispensable+for+hematopoietic+stem+cell+mobilization+induced+by+interleukin-8+in+mice.">Neutrophils are indispensable for hematopoietic stem cell mobilization induced by interleukin-8 in mice.</a> Proc Natl Acad Sci U S A. 99, 6228-6233 (2002).</li><li>Tecchio, C., 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=Neutrophil-derived+cytokines+involved+in+physiological+and+pathological+angiogenesis.">Neutrophil-derived cytokines involved in physiological and pathological angiogenesis.</a> Chem Immunol Allergy. 99, 123-137 (2014).</li><li>Liu, 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=Formylpeptide+receptors+mediate+rapid+neutrophil+mobilization+to+accelerate+wound+healing.">Formylpeptide receptors mediate rapid neutrophil mobilization to accelerate wound healing.</a> PloS one. 9, e90613 (2014).</li><li>Granot, Z., 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=Tumor+entrained+neutrophils+inhibit+seeding+in+the+premetastatic+lung.">Tumor entrained neutrophils inhibit seeding in the premetastatic lung.</a> Cancer Cell. 20, 300-314 (2011).</li><li>Sionov, R. V., Fridlender, Z. G., Granot, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Multifaceted+Roles+Neutrophils+Play+in+the+Tumor+Microenvironment.">The Multifaceted Roles Neutrophils Play in the Tumor Microenvironment.</a> Cancer Microenviron. , (2014).</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>Yamashiro, 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=Phenotypic+and+functional+change+of+cytokine-activated+neutrophils:+inflammatory+neutrophils+are+heterogeneous+and+enhance+adaptive+immune+responses.">Phenotypic and functional change of cytokine-activated neutrophils: inflammatory neutrophils are heterogeneous and enhance adaptive immune responses.</a> J Leukoc Biol. 69, 698-704 (2001).</li><li>Futosi, K., Fodor, S., Mocsai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+cell+surface+receptors+and+their+intracellular+signal+transduction+pathways.">Neutrophil cell surface receptors and their intracellular signal transduction pathways.</a> Int Immunopharmacol. 17, 638-650 (2013).</li><li>Scapini, P., 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=Social+networking+of+human+neutrophils+within+the+immune+system.">Social networking of human neutrophils within the immune system.</a> Blood. 124, 710-719 (2014).</li><li>Fridlender, Z. G., 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=Transcriptomic+analysis+comparing+tumor-associated+neutrophils+with+granulocytic+myeloid-derived+suppressor+cells+and+normal+neutrophils.">Transcriptomic analysis comparing tumor-associated neutrophils with granulocytic myeloid-derived suppressor cells and normal neutrophils.</a> PloS one. 7, e31524 (2012).</li><li>Fridlender, Z. G., 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=Polarization+of+tumor-associated+neutrophil+phenotype+by+TGF-beta:+'N1'+versus+'N2.">Polarization of tumor-associated neutrophil phenotype by TGF-beta: 'N1' versus 'N2.</a> TAN. Cancer Cell. 16, 183-194 (2009).</li><li>Talmadge, J. E., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+myeloid-derived+suppressor+cells.">History of myeloid-derived suppressor cells.</a> Nat Rev Cancer. 13, 739-752 (2013).</li><li>Youn, J. I., Nagaraj, S., Collazo, M., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsets+of+myeloid-derived+suppressor+cells+in+tumor-bearing+mice.">Subsets of myeloid-derived suppressor cells in tumor-bearing mice.</a> J Immunol. 181, 5791-5802 (2008).</li><li>Choi, 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=CD15+/CD16low+human+granulocytes+from+terminal+cancer+patients:+granulocytic+myeloid-derived+suppressor+cells+that+have+suppressive+function.">CD15+/CD16low human granulocytes from terminal cancer patients: granulocytic myeloid-derived suppressor cells that have suppressive function.</a> Tumour Biol. 33, 121-129 (2012).</li><li>Brandau, 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=Myeloid-derived+suppressor+cells+in+the+peripheral+blood+of+cancer+patients+contain+a+subset+of+immature+neutrophils+with+impaired+migratory+properties.">Myeloid-derived suppressor cells in the peripheral blood of cancer patients contain a subset of immature neutrophils with impaired migratory properties.</a> J Leukoc Biol. 89, 311-317 (2011).</li><li>Pillay, J., Tak, T., Kamp, V. M., Koenderman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+suppression+by+neutrophils+and+granulocytic+myeloid-derived+suppressor+cells:+similarities+and+differences.">Immune suppression by neutrophils and granulocytic myeloid-derived suppressor cells: similarities and differences.</a> Cell Mol Life Sci. 70, 3813-3827 (2013).</li><li>Lopez-Lago, M. A., 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=Neutrophil+chemokines+secreted+by+tumor+cells+mount+a+lung+antimetastatic+response+during+renal+cell+carcinoma+progression.">Neutrophil chemokines secreted by tumor cells mount a lung antimetastatic response during renal cell carcinoma progression.</a> Oncogene. 32, 1752-1760 (2013).</li><li>Boxio, R., Bossenmeyer-Pourie, C., Steinckwich, N., Dournon, C., Nusse, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+bone+marrow+contains+large+numbers+of+functionally+competent+neutrophils.">Mouse bone marrow contains large numbers of functionally competent neutrophils.</a> J Leukoc Biol. 75, 604-611 (2004).</li><li>Sagiv, 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=Phenotypic+Diversity+and+Plasticity+in+Circulating+Neutrophil+Subpopulations+in+Cancer.">Phenotypic Diversity and Plasticity in Circulating Neutrophil Subpopulations in Cancer.</a> Cell Reports. , (2015).</li></ol>

The authors have nothing to disclose.

好中球は、すべての白血球の最も豊富であり、感染症や炎症の場合には最初の対応です。そのようなものとして、それらは、外部の合図に非常に敏感であり、容易に活性化されます。また、好中球は非常に短い半減期および迅速なターンオーバーを持っています。一緒に、これらの特性は、ユニークな実験的な戦略が必要とされるように、好中球での作業のいくつかの困難を、上げます。例?...

好中球は、最初に侵入微生物に対する最初の行の防御として機能する自然免疫細胞として特徴づけられました。今日では、好中球は造血3の調節、外来抗原1,2-に対する適応免疫応答をマウントに関与している多くの広範囲の機能を有することが知られている血管新生4,5創傷治癒します。さらに、好中球は、それらのプロ及び抗腫瘍活性6,7によって腫瘍の増殖および転移の進行に影響を及ぼし得ます。好中球は、多型、セグメント化、核によって特徴付けられる（したがって、多形核（PMN）白血球と呼ばれる）と、少なくとも3顆粒の別個のサブクラスならびに分泌小胞8（ 図1A-C）を含有します。 好中球は、高い貪食能と微生物の除去のための重要な高NADPHオキシダーゼ活性を有しており、時のための重要なケモカインの広い範囲を分泌します炎症8,9の部位に付加的な好中球および他の免疫細胞の牽引。好中球は、Toll様受容体（TLR）を含む表面受容体の多量の発現を特徴とする、C型レクチン受容体（のCLR）は、受容体3（のCD11b / CD18）、および他の接着分子（ 例えば、L-セレクチンを補完しますLFA-1、VLA-4および癌胎児性抗原関連細胞接着分子3（CEACAM3 / CD66b））、ケモカイン受容体（ 例えば、CXCR1、CXCR2、CCR1、CCR2）、化学誘引物質受容体（ 例えば、PAFR、LTB 4 RとのC5aR） 、サイトカイン受容体（ 例えば、G-CSFR、IL-1R、IL-4R、IL-12R、IL-18R、TNFR）、ホルミルペプチド受容体（ 例えば、FPR1-3）、およびFc受容体（ 例えば、CD16（FcγRIIIをヒト好中球はのCD11b、CD15、CD16およびCD66b白血球マーカーを用いて同定されるのに対して）、CD32（のFcγRII）およびCD64（FcγRIで）10。マウスでは、好中球は通常、のCD11b + Ly6G +として同定されています。また、一般的に組織中の好中球の検出のための顆粒タンパク質のミエロペルオキシダーゼ（MPO）と好中球エラスターゼ（NE）のために染色する受け入れられています。 これは、好中球の多様な機能が同じ細胞により、または異なる細胞亜集団によって媒介されるかどうかはまだ不明です。蓄積データは、プロ炎症性刺激と微小環境11,12の影響を受けて可塑性の高度を示す異種好中球集団の存在を示唆しています。 Fridlender ら 13は、2つの主要な亜集団に癌において好中球を著しく分割しているプロ腫瘍特性を有する抗腫瘍特性を有するN1およびN2と呼ばれます。癌において、ならびに慢性炎症において、T細胞応答14を抑制顆粒骨髄由来サプレッサー細胞（G-たMDSC）からなる追加の亜集団が存在します。 G-のMDSCはのCD11b + Ly6Cによって特徴付け未熟骨髄細胞であると考えられていますマウス15で低 Ly6G こんにちは表現型、ヒト16でCD15 + / CD16 低の表現型を有しながら。 G-たMDSC通常よりもサイトカインおよびケモカインのより低いレベルは、好中球を循環させながら、アルギナーゼおよびミエロペルオキシダーゼのより高いレベルを発現します。彼らはあまり食作用および移動であるが、ROS 15,17,18の高いレベルを生成します。本論文では、抗腫瘍特性を有する好中球の単離および特徴付けのためにいくつかの基本的な方法論を説明します。 （45から70までパーセント; 1,800 - 6,000 /μl）を好中球は、ヒトの循環内のすべての白血球の最大の人口を構成しているが、マウスでは、通常の条件下で、彼らは（10むしろ疎である - 15％、300から500 /μL ）。慢性炎症7の状態を示して着実に炎症時に時折癌における好中球数が増加します。好中球は多能骨髄系共通前駆体（CMP）CEから発生します骨髄中のLLS、骨髄芽球の段階を通過する分化プロセス（MB）を介して、前骨髄球（PM）、骨髄球（MC）、後骨髄球（MM）とバンド細胞（BC）8。それらは循環8に放出される前に7日-成熟した、有糸分裂後の好中球は4のための骨髄内に残ることがあります。炎症状態の下で延長することができる12時間、 - 血液中の好中球の売上高は6の平均半減期は、通常、迅速です。非刺激好中球は、ホルボールエステルホルボール12にそれらを暴露することにより、抗腫瘍形成活性、ケモカインIL-8（CXCL2）、CCL2、CCL5およびCXCL5 6,19または人工的にナイーブな好中球を露出することによって取得することができる機能を制限してきました-myristate 13-アセテート（PMA）6。 一緒に好中球の数が少ないと血液の好中球の半減期が短い（〜3から5×10 5）ナイーブ6 1mlの血液から達成- 8週齢のマウス、それを作りましたin vitroでマウスの好中球を循環させる機能を探索することは困難。この困難を克服するために、他のソースが使用されています。例えば、好中球の多くは、（チオグリコレート培地またはザイモサンAの腹腔内注射後に、 例えば ）骨髄20または無菌性炎症の誘導後腹膜から得ることができます。これは、腹腔から得られた好中球は、任意の抗腫瘍形成活性（未発表の観察）を及ぼさないことに留意すべきです。 Granot ら 6 20れるよう、マウス4T1乳癌細胞株を用いて同所接種したBALB / cマウスは、腫瘍進行6（ 図2A）で悪化好中球増加症を発症することが観察- 40万人の血液好中球は容易に1ミリリットルの血液から単離することができます3から4週間後、腫瘍接種。これらの好中球は、抗腫瘍活性を獲得し、それに応じてCOIされていますナイーブ好中球6（ 図2B）と区別するために定義される腫瘍連行好中球（TEN）、。高密度の好中球（HDN、 図1（a））は非常に抗腫瘍形成であるが、癌との関連で発生した低密度の好中球（LDN、 図1B）は 21ではありません。また、腫瘍を有するマウスの骨髄および脾臓から高密度の好中球は、抗腫瘍活性（未発表データ）を有しています。これは、好中球の量が増加すると、腫瘍の進行に脾臓を徐々に拡大（脾腫）となることに留意すべきです。 これは、10にも自発的な（MMTV-PyMTおよびMMTV-Wnt1の乳腺腫瘍とのK-ras駆動肺腫瘍）の両方を含む癌の他のモデルで生成され、AT-3（MMTV-PyMT）とE0771の乳癌（注入されることに留意すべきです細胞、LLCルイス肺癌細胞およびB16-F10黒色腫細胞）。しかしながら、これらのTUMでの好中球動員の、程度3週間後に1ミリリットルの血液中の10×10 6好中球-またはモデルが5に達し、4T1-接種されたマウスのよりもはるかに少ないです。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
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			<td>CELL LINES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse 4T1 breast carcinoma cells</td>
			<td>ADCC</td>
			<td>CRL-2539</td>
			<td>Growth medium: DMEM + 10 % heat-inactivated FBS</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PLASTIC WARES AND EQUIPMENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Tissue Culture Plate&#160;</td>
			<td>Falcon</td>
			<td>353047</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>100 mm Tissue Culture Plate&#160;</td>
			<td>Corning</td>
			<td>430167</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>25 cm2 Tissue Culture Flask</td>
			<td>Nunc</td>
			<td>156340</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>90 mm Bacterial Grade Culture Dish&#160;</td>
			<td>Miniplast, Ein Shemer, Israel</td>
			<td>20090-01-017</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>15 ml Sterile Conical Centrifuge Tube&#160;</td>
			<td>Miniplast, Ein Shemer, Israel</td>
			<td>835015-40-111</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>50 ml Sterile Conical Centrifuge Tube&#160;</td>
			<td>Miniplast, Ein Shemer, Israel</td>
			<td>835050-21-111</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Falcon 12x75 mm Round-Bottom Polystyrene Tube&#160;</td>
			<td>Becton Dickinson</td>
			<td>352058</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Millicell 24 Migration Plate with a pore size of 5&#956;m&#160;</td>
			<td>Merck Millipore</td>
			<td>PSMT010R1</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>White 96-Flat-Bottom Well Plate&#160;</td>
			<td>Costar</td>
			<td>3917</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Cell Strainer (40 mm)&#160;</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>20G 1.5&#34; Needle</td>
			<td>BD Microlance 3&#160;</td>
			<td>301300</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>23G 1&#34; Needle&#160;</td>
			<td>BD Microlance 4</td>
			<td>300800</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>25Gx5/8&#34; Needle&#160;</td>
			<td>BD Microlance 5</td>
			<td>300600</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>0.3 ml Syringe with a 30Gx8mm Needle</td>
			<td>BD Micro-Fine Plus Demi</td>
			<td>320829</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>9 mm Clips&#160;</td>
			<td>BD, AutoClip&#160;</td>
			<td>427631</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>EasySep Magnet&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS LS Separation Column&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-201</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>MidiMACS Separator Magnet</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Glass Slide&#160;</td>
			<td>Menzel-Gl&#228;ser Superfrost&#160; Plus Thermo</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker&#160;</td>
			<td>Sky line, ELMI</td>
			<td>S-3.02.10L</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Reader&#160;</td>
			<td>TECAN</td>
			<td>InfiniteF200Pro</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>POWDER</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA), fraction V</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine (BrdU)&#160;</td>
			<td>BD Pharmingen</td>
			<td>550891</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>CFSE (5-(and 6-)-Carboxyfluorescein diacetate, succinimidyl ester)</td>
			<td>Molecular Probes</td>
			<td>C1157</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran T500</td>
			<td>Sigma</td>
			<td>31392</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa&#160;</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide (NaN3)</td>
			<td>Sigma</td>
			<td>S8032</td>
			<td>Highly toxic, handle with care</td>
		</tr>
		<tr>
			<td>Thioglycollate powder&#160;</td>
			<td>Difco</td>
			<td>225650</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymosan A</td>
			<td>Sigma</td>
			<td>Z4250</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEDIA AND SUPPLEMENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle medium (DMEM)</td>
			<td>Sigma</td>
			<td>D5796</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Opti-MEM&#174; I reduced serum medium&#160;</td>
			<td>Life Technologies</td>
			<td>31985062</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI)-1640 medium</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Foetal bovine serum (FBS), heat-inactivated</td>
			<td>Sigma</td>
			<td>F9665</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-020-1A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-042-1B</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin x1000 solution</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-031-5</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) without Mg2+ and Ca2+&#160;</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>02-023-1</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>PBSx10 without Ca2+ and Mg2+&#160;</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>02-023-5A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>HPLC grade water&#160;</td>
			<td>J.T. Baker</td>
			<td>4218-03</td>
			<td>Autoclave</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SOLUTIONS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACK &#8211; Ammonium-Chloride-Potassium</td>
			<td>Life Technologies&#160;</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine (BrdU) solution (10 mg/ml) in PBS</td>
			<td></td>
			<td></td>
			<td>Dissolve 10 mg of BrdU in 1 ml PBS and sterile filter.</td>
		</tr>
		<tr>
			<td>CFSE, 5 mM in DMSO</td>
			<td></td>
			<td></td>
			<td>Dissolve 2.8 mg of CFSE in 1 ml DMSO. Divide into 10 ml aliquots in sterile 200 ml tubes and store in the dark at -20oC.</td>
		</tr>
		<tr>
			<td>Eosin Y solution</td>
			<td>Sigma</td>
			<td>HT110-2-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; balanced salt solution</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>02-016-1A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Heparin, 20 mg/ml in PBS</td>
			<td></td>
			<td></td>
			<td>Dissolve 100 mg Heparin in 5 ml sterile PBS, and sterile filter through a 0.2 mm filter.&#160;</td>
		</tr>
		<tr>
			<td>Histopaque-1119&#160;</td>
			<td>Sigma</td>
			<td>11191</td>
			<td>Sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>Histopaque-1077&#160;</td>
			<td>Sigma</td>
			<td>10771</td>
			<td>Sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>&#160;Luciferase cell culture lysis buffer x5</td>
			<td>Promega</td>
			<td>E153A</td>
			<td>Dilute 1:5 in sterile water just before use.</td>
		</tr>
		<tr>
			<td>Luciferase assay solution</td>
			<td>Promega</td>
			<td>E1501</td>
			<td>Contains luciferase assay substrate powder (E151A) and luciferase assay buffer (E152A)</td>
		</tr>
		<tr>
			<td>Mayer&#39;s Hematoxylin solution&#160;</td>
			<td>Sigma</td>
			<td>MHS-32</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS+0.5% BSA</td>
			<td></td>
			<td></td>
			<td>Dissolve 2.5g BSA in 500 ml PBS, and sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>PBS+1% BSA</td>
			<td></td>
			<td></td>
			<td>Dissolve 1g BSA in 100 ml PBS, and sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>5x PBS with 2.5% BSA</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 12.5g BSA in a mixture of 250 ml sterile HPLC-grade water&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; and 250 ml PBSx10, and sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>PBS containing 0.5% BSA and 2 mM EDTA</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 250 mg BSA in 50 ml sterile PBS&#160; and add 200 ml of 0.5M EDTA pH 8.0, sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>FACS buffer (PBS containing 0.5% BSA, 2 mM EDTA and 0.02% NaN3)</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 250 mg BSA in 50 ml sterile PBS&#160; and add 200 ml of 0.5M EDTA pH 8.0 and 500 ml of 2% NaN3, sterile filter through a 0.2 mm filter.</td>
		</tr>
		<tr>
			<td>Saline (0.9% NaCl)</td>
			<td></td>
			<td></td>
			<td>Dissolve 9 g NaCl in 1000 ml ddw, autoclave</td>
		</tr>
		<tr>
			<td>0.2% NaCl solution</td>
			<td></td>
			<td></td>
			<td>Dissolve 2 g NaCl in 1000 ml ddw, autoclave</td>
		</tr>
		<tr>
			<td>1.6% NaCl solution</td>
			<td></td>
			<td></td>
			<td>Dissolve 16 g NaCl in 1000 ml ddw, autoclave</td>
		</tr>
		<tr>
			<td>2 % Sodium azide</td>
			<td></td>
			<td></td>
			<td>Dissolve 1g sodium azide in 50 ml sterile ddw, keep at 4oC. Highly toxic.</td>
		</tr>
		<tr>
			<td>3% Thioglycollate solution</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 3 g of thioglycollate powder in 100 ml ddw.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Boil until solution becomes yellow and autoclave.</td>
		</tr>
		<tr>
			<td>Trypan blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>Dilute 1:10 in PBS to get a 0.04% solution.</td>
		</tr>
		<tr>
			<td>Trypsin solution B&#160;</td>
			<td>Biological Industries, Beth HaEmek, Israel</td>
			<td>03-046-1</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>1 mg/ml Zymosan A</td>
			<td></td>
			<td></td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Resuspend 1 mg Zymosan A&#160; in 1 ml sterile PBS in an Eppendorf tube.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Vortex vigorously and incubate the tube at 37 oC for 30 min. Do not autoclave.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Prepare the solution freshly before use.&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KITS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep PE selection kit</td>
			<td>STEMCELL Technologies</td>
			<td>18557</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep PE selection cocktail&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18151</td>
			<td></td>
		</tr>
		<tr>
			<td>the EasySep magnetic nanoparticles&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18150</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ly6G mouse MicroBead Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-332</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Neutrophil Enrichment Kit</td>
			<td>STEMCELL Technologies</td>
			<td>19762</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human Neutrophil Enrichment Kit</td>
			<td>STEMCELL Technologies</td>
			<td>19257</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC BrdU flow kit</td>
			<td>BD Pharmingen&#160;</td>
			<td>559619</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Neutrophil isolation kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Phagocytosis Assay Kit&#160;</td>
			<td>Cayman Chemical Company&#160;</td>
			<td>500290</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ANTIBODIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FcR blocking antibody&#160;</td>
			<td>Biolegend</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified rat anti-Ly6G antibody&#160;</td>
			<td>BD Pharmingen&#160;</td>
			<td>551459</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>PE-conjugated rat anti-mouse Ly6G antibody&#160;</td>
			<td>Biolegend</td>
			<td>127608</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse Ly6G</td>
			<td>BD Pharmingen&#160;</td>
			<td>551460</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5 rat anti-mouse Ly6G&#160;</td>
			<td>TONBO Biosciences</td>
			<td>65-1276</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>violetFluor 450-conjugated rat anti-mouse Ly6G&#160;</td>
			<td>TONBO Biosciences</td>
			<td>75-1276</td>
			<td>Clone 1A8</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse CD11b</td>
			<td>BD Pharmingen&#160;</td>
			<td>553310</td>
			<td>Clone M1/70</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse Ly-6G and Ly-6C (GR-1)</td>
			<td>BD Pharmingen&#160;</td>
			<td>553127</td>
			<td>Clone RB6-8C5</td>
		</tr>
		<tr>
			<td>PE-conjugated rat anti-mouse CD45</td>
			<td>BD Pharmingen&#160;</td>
			<td>553081</td>
			<td>Clone 30-F11</td>
		</tr>
		<tr>
			<td>FITC-conjugated rat anti-mouse F4/80&#160;</td>
			<td>Abcam</td>
			<td>ab60343</td>
			<td>Clone BM8</td>
		</tr>
		<tr>
			<td>FITC-conjugated mouse anti-human CD66b&#160;</td>
			<td>Biolegend</td>
			<td>305103</td>
			<td>Clone G10F5</td>
		</tr>
		<tr>
			<td>Purified rat isotype control antibody (IgG2a, k)&#160;</td>
			<td>BD Pharmingen&#160;</td>
			<td>553927</td>
			<td>Clone R35-95</td>
		</tr>
		<tr>
			<td>LEAF purified Armenian hamster anti-mouse CD3e antibody</td>
			<td>BioLegend</td>
			<td>100314</td>
			<td>Clone 145-2C11</td>
		</tr>
	</tbody>
</table>

isolation and characterization of neutrophils with anti-tumor properties

好中球は、ヒト循環中の全白血球の最も豊富な、微生物侵入に対する宿主防御において重要な役割を果たしています。さらに、好中球は、腫瘍細胞の免疫監視において中心的な役割を果たす。彼らは、腫瘍細胞を認識し、細胞接触依存性機構含む過酸化水素を介して、または抗体依存性細胞媒介性細胞傷害（ADCC）を介してのいずれかで腫瘍細胞死を誘導する能力を有します。抗腫瘍活性を有する好中球は、癌患者の腫瘍を有するマウスの末梢血から単離することができます。これらの好中球は、有意な腫瘍細胞傷害活性を示さない健常者またはナイーブマウスの好中球と区別するために、腫瘍同伴好中球（TEN）と呼ばれています。他の白血球と比較して、好中球は、密度勾配にかけたときに可能&gt; 98％純粋な好中球の集団を得ることができる別の浮力を示しています。しかし、加えて、通常高密度の好中球集団（HDN）に、癌患者において、腫瘍を有するマウスにおける、ならびに慢性炎症性条件下で、明確な低密度好中球集団（LDN）は、循環に表示されます。 LDNは、単核画分と共精製すると、正または負の選択戦略を用いて、単核細胞から分離することができます。単離された好中球の純度は、フローサイトメトリーにより決定されると、それらは、 インビトロおよびインビボ機能アッセイにおいて用いることができます。我々は、好中球の抗腫瘍活性を監視するための能力移行すると活性酸素種を生成すること、ならびにそれらの貪食能のex vivoでモニタリングする技術を説明します。我々はさらに、in vivoで追跡するための好中球を標識するための技術を説明し、そしてインビボでの抗転移能力を決定します。すべてのこれらの技術は、抗腫瘍で好中球を取得し、特徴づけるためにどのように理解するために不可欠です機能。

最近の研究では、好中球6のための抗転移作用を同定しました。腫瘍を有するマウスからの好中球は、細胞傷害性表現型を獲得し、腫瘍細胞の6を殺す能力を有します。これは、有意な抗腫瘍効果6を有していないナイーブマウスからの好中球とは対照的です。プロトコルセクションに記載された技術のいくつかは、in vitroおよびin vivo 6 において抗?...

Watch this Scientific Journal Video about Isolation and Characterization of Neutrophils with Anti-Tumor Properties at JoVE.com

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

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 ...

isolation-characterization-neutrophils-with-anti-tumor

Research

JoVE Journal

Immunology and Infection

24.6K ビュー.  Hebrew University Medical School. 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. 

ビデオ: Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

ウイルスの変異頻度のRNAウイルスと特性の忠実性の変異の分離

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

免疫・感染学

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

マウスの腸からの樹状細胞およびマクロファージの単離とキャラクタリゼーション

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎球菌による鼻腔内コロニー形成時の炎症反応の特徴

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

機能研究のためのマウス骨髄好中球と養子移入実験の分離、精製とラベリング

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

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. ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

単離、特徴付けおよび歯肉免疫細胞ネットワークの機能検討

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

マウスの小腸リンパ球の単離およびフローサイトメトリーキャラクタリゼーション

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用してリンパ球血管外漏出の分析<em&gt;インビトロ</em&gt;ヒト血液脳関門のモデル

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

分離と機能研究の好中球由来の微粒子のキャラクタリゼーション

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

腹膜由来マスト細胞とその機能特性と Ca2 +の単離-イメージングと脱顆粒の試金

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...