私たちは、優れた技術支援のためナディーンのWambsganssに感謝。この作品は、CAGにイノベーション基金FRONTIER（ハイデルベルク大学）からの助成金によってドイツ学術振興（GL599/1-1）からの助成金によって部分的にサポートされていました

1。プロトコル<ol><li>次のようにバッファを準備します。 <ol><li> PBMCの分離のためのバッファを準備します。PBS中= 0.02％EDTAを（使用0.5 M EDTA） &quot;バッファを洗う&quot;。 </li><li>単離のためにバッファを準備します。 &quot;MACSすすぎバッファ&quot; = 0.5％BSA（250 mg）を+ 2mMのEDTA（200μlの）+ PBS（50ミリリットル）。ドガバッファ。 </li><li> &quot;FACSバッファー&quot; PBS中= 10％FCSと &quot;固定バッファ&quot; PBS中= 1％PFA：細胞のFACS染色および保管するためのバッファを準備します。 </li></ol></li><li>前腕静脈から30ミリリットルの全血を引く。抗凝固剤としてEDTAを使用してください。 </li><li>次のようにPBMC（HISTOPAQUE）を単離： <ol><li> 2無菌の50mlチューブ（無ポリ - スチレン）を準備します。 </li><li> PBS 1:1でステップ2から血液を希釈します。 </li><li> + 25ミリリットルの全血/ PBSチューブあたり25ミリリットルのHISTOPAQUEを追加します。 HISTOPAQUEと血が混ざらないことを確認してください。 </li><li> RT、ブレーキなしで30分間400×gで遠心分離します。 </li><li>血漿および廃棄を吸引。 </li><li>クリーンの50ml浴槽に不透明なインターフェイスとピペットを吸引電子。 </li><li> PBS中0.02％EDTAを20mlを加える。 </li><li> 4℃で5分間、250×gで遠心</li><li>上清を捨てます。 </li><li> PBS中0.02％EDTAを10mlを加える。 </li></ol></li><li>次のようにセルを数える。 <ol><li> 10秒間ボルテックスでよく混ぜる。 </li><li> 200μlの細胞懸濁液+ 300μlのPBS中0.02％EDTA +トリパンブルー（200-500 cells/10正方形、そうでない希釈率を変更します）の500μLを取る。 </li></ol></li><li>次のように単球の負の分離を実行します。 <ol><li> 10分、120×gのためにステップ3.10で得られた細胞を遠心分離。上清を捨てます。 </li><li> 10 mlのPBS + 0.02％EDTA、10分間、120×gで遠心を追加することで、細胞ペレット2倍を洗ってください。 </li><li> 1ミリリットル10X PBSを加え、3秒間9ミリリットル滅菌水を追加します。 </li><li> PBS + 0.02％EDTAでもう一度洗浄し、10分間、120×gで遠心。 </li><li>遠心分離中数える。 </li><li> 5×10 7 / mlでEasySepバッファーに希釈します。 </li><li> 50ミリリットル/μlの単球濃縮コックを追加尾。 </li><li> 4℃で10分インキュベート</li><li> 30秒間ボルテックスビーズ。 </li><li> 50μL/ mlのビーズを追加します。 </li><li> 4℃で5分間インキュベート</li><li> 2.5ミリリットルの容積を完了するEasySepバッファーでいっぱい。解決策は今茶色がかった表示されます。 </li><li>磁石に入れ。 </li><li>室温で2.5分待ちます。この間磁気ビーズに結合した非単球細胞が管壁に移動し、そこに固執する。 </li><li>フレッシュ無菌チューブに非結合単球とバッファを注ぐ。このソリューションでは白っぽく見えます。 </li><li> PBS + 0.02％EDTAで一回洗浄し、10分間、120×gで遠心します。 </li></ol></li><li> 0.5×10 6個/ cm 2の密度でプラスチックの皿または多層プレートのプレートの細胞。培地/ 1×10 6個の細胞を1 mlを加える。 </li><li> 6-14日間興味のある条件下での培養細胞。 </li><li> （詳細については表1を参照）は、新鮮なメディアでのメディアの50％を置き換えることにより、3日後にメディアを変更してください。 </li><li>後の6日は、mRNAの分離、フローサイトメトリー、ウェスタンブロット法、 等のための細胞を回収。 </li></ol>

<ol>
	<li>Gordon, S., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+and+macrophage+heterogeneity.">Monocyte and macrophage heterogeneity.</a> Nat. Rev. Immunol. 5, 953-964 (2005).</li><li>Gordon, S., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+plasticity+of+mononuclear+phagocytes.">Diversity and plasticity of mononuclear phagocytes.</a> European Journal of Immunology. 41, 2470-2472 (2011).</li><li>Martinez, F. O., Sica, A., Mantovani, A., Locati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+and+polarization.">Macrophage activation and polarization.</a> Front. Biosci. 13, 453-461 (2008).</li><li>Ingersoll, 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=Comparison+of+gene+expression+profiles+between+human+and+mouse+monocyte+subsets.">Comparison of gene expression profiles between human and mouse monocyte subsets.</a> Blood. 115, 10-19 (2010).</li><li>Gleissner, C. A., Sanders, J. M., Nadler, J., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+aldose+reductase+during+foam+cell+formation+as+possible+link+among+diabetes,+hyperlipidemia,+and+atherosclerosis.">Upregulation of aldose reductase during foam cell formation as possible link among diabetes, hyperlipidemia, and atherosclerosis.</a> Arterioscler. Thromb. Vasc. Biol. 28, 1137-1143 (2008).</li><li>Gleissner, C. 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=CXCL4+downregulates+the+atheroprotective+hemoglobin+receptor+CD163+in+human+macrophages.">CXCL4 downregulates the atheroprotective hemoglobin receptor CD163 in human macrophages.</a> Circ. Res. , (2009).</li><li>Gleissner, C. A., Shaked, I., Little, K. M., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXC+chemokine+ligand+4+induces+a+unique+transcriptome+in+monocyte-derived+macrophages.">CXC chemokine ligand 4 induces a unique transcriptome in monocyte-derived macrophages.</a> J. Immunol. 184, 4810-4818 (2010).</li><li>Martinez, F. O., Gordon, S., Locati, M., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+the+human+monocyte-to-macrophage+differentiation+and+polarization:+New+molecules+and+patterns+of+gene+expression.">Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: New molecules and patterns of gene expression.</a> J. Immunol. 177, 7303-7311 (2006).</li><li>Verreck, F. A. W., 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=Human+il-23-producing+type+1+macrophages+promote+but+il-10-producing+type+2+macrophages+subvert+immunity+to+(myco)bacteria.">Human il-23-producing type 1 macrophages promote but il-10-producing type 2 macrophages subvert immunity to (myco)bacteria.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 4560-4565 (2004).</li><li>Rey-Giraud, F., Hafner, M., Ries, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+generation+of+monocyte-derived+macrophages+under+serum-free+conditions+improves+their+tumor+promoting+functions.">In vitro generation of monocyte-derived macrophages under serum-free conditions improves their tumor promoting functions.</a> PLoS One. 7, e42656 (2012).</li><li>Boyle, J. 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=Coronary+intraplaque+hemorrhage+evokes+a+novel+atheroprotective+macrophage+phenotype.">Coronary intraplaque hemorrhage evokes a novel atheroprotective macrophage phenotype.</a> American Journal of Pathology. 174, 1097-1108 (2009).</li><li>Cho, H. 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=Induction+of+dendritic+cell-like+phenotype+in+macrophages+during+foam+cell+formation.">Induction of dendritic cell-like phenotype in macrophages during foam cell formation.</a> Physiol Genomics. 29, 149-160 (2007).</li><li>Vogt, G., Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+human+macrophages+with+enhanced+antimycobacterial+activity.">In vitro differentiation of human macrophages with enhanced antimycobacterial activity.</a> The Journal of Clinical Investigation. 121, 3889-3901 (2011).</li><li>Mikita, T., Porter, G., Lawn, R. M., Shiffman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidized+low+density+lipoprotein+exposure+alters+the+transcriptional+response+of+macrophages+to+inflammatory+stimulus.">Oxidized low density lipoprotein exposure alters the transcriptional response of macrophages to inflammatory stimulus.</a> Journal of Biological Chemistry. 276, 45729-45739 (2001).</li><li>Qin, Z. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+THP-1+cells+as+a+model+for+mimicking+the+function+and+regulation+of+monocytes+and+macrophages+in+the+vasculature.">The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature.</a> Atherosclerosis. 221, 2-11 (2012).</li><li>Daigneault, M., Preston, J. A., Marriott, H. M., Whyte, M. K. B., Dockrell, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+of+markers+of+macrophage+differentiation+in+pma-stimulated+THP-1+cells+and+monocyte-derived+macrophages.">The identification of markers of macrophage differentiation in pma-stimulated THP-1 cells and monocyte-derived macrophages.</a> PLoS One. 5, e8668 (2010).</li><li>Wahl, L. M., Wahl, S. M., Smythies, L. E., Smith, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+monocyte+populations.">Isolation of human monocyte populations.</a> Current Protocols in Immunology. , (2001).</li><li>Waldo, S. W., 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=Heterogeneity+of+human+macrophages+in+culture+and+in+atherosclerotic+plaques.">Heterogeneity of human macrophages in culture and in atherosclerotic plaques.</a> Am. J. Pathol. 172, 1112-1126 (2008).</li><li>Mart&#237;n-Fuentes, 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=Individual+variation+of+scavenger+receptor+expression+in+human+macrophages+with+oxidized+low-density+lipoprotein+is+associated+with+a+differential+inflammatory+response.">Individual variation of scavenger receptor expression in human macrophages with oxidized low-density lipoprotein is associated with a differential inflammatory response.</a> The Journal of Immunology. 179, 3242-3248 (2007).</li></ol>

著者は利益相反を開示する必要はありません。

単球由来マクロファージは自然免疫系の主要な細胞型を表す。それらは、アテローム性動脈硬化症または癌2を含む多くの炎症性疾患において重要な役割を果たす。したがって、マクロファージ生物学を研究することは、多くの疾患の病態生理に知識を増大させるために不可欠であり、新規な治療法の開発を可能にする。 多くの研究は、マウスモデルの過剰発現?...

単球由来のマクロファージは自然免疫系の重要な細胞成分を表し、多くの急性または慢性炎症プロセス1に貢献しています。マクロファージは、アテローム性動脈硬化症や癌2のような多くの炎症性疾患において重要な役割を果たしている。マクロファージは、可塑性の高度を示し、局所微小環境3に応じて、異なる表現型を想定することができます。したがって、マクロファージの分化と異質性を研究することは、多くの疾患の病態生理学の知識を増大させるために不可欠であり、新たな治療標的とする新規治療法の開発の識別を可能にする。 多くの場合、マウスモデルは、特定の疾患の病態を研究するために使用される。しかし、マウスモデルを用いたマクロファージ生物学を勉強するいくつかの欠点が付属しています：（1）peripにおける白血球サブセット数の割合（ すなわち、単球および顆粒球）マウスやヒトのheral血がかなりマウスおよびヒトの病態生理における単球のさまざまな役割を示唆しているとは異なります。 （2）健康および疾患4の間に、それらの機能の実質的な差異を示唆するマウスおよびヒト末梢血単核球との間の遺伝子発現の実質的な違いがある。 （3）マウス単球およびマクロファージを識別するために使用されるマーカーの数（F4/80、LYC など ）、ヒト状況にマウスモデルにおいて調査結果の転送はむしろ困難になる、ヒト骨髄細胞には存在しない。 したがって、ヒトの疾患におけるマクロファージの分化と異質の理解を高めるために、我々は、ヒトマクロファージでの作業モデルを利用する必要がある。したがって、我々は、ここで使用することは簡単ですし、別のマクロファージPOの結果、様々な条件下でのin vitroでのヒト単球由来マクロファージの研究を可能にするヒト初代マクロファージ世代のモデルを記述larizationタイプ。いくつかの研究において、我々は、マクロファージ生物学および5-7ヒトアテローム性動脈硬化症への潜在的な関連性を分析するために単球由来の初代ヒトマクロファージのin vitroモデルにおいて使用している。 にもかかわらず、完全に死後得動物やヒト組織を用いた実験を交換しない、ここで説明するモデルは、病気のメカニズムや様々なヒト疾患との関連性が高いかもしれない治療標的の同定と検証を可能にします。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube (sterile)</td>
			<td>Fisher</td>
			<td>055398</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-PBS (1X), liquid (no calcium or magnesium)</td>
			<td>Invitrogen</td>
			<td>14190-250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>T9285</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-8806</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EasySep Human Monocyte Enrichment Kit</td>
			<td>StemCell Technologies</td>
			<td>19059</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fisher</td>
			<td>352008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Macrophage-SFM (1X)</td>
			<td>Invitrogen</td>
			<td>12065-074</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P-4458</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nutridoma-SP</td>
			<td>Roche</td>
			<td>11011375001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>human M-CSF 10 &mu;g</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell Culture Plates 6-well</td>
			<td>Fisher</td>
			<td>07-200-80</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an in vitro model to study heterogeneity of human macrophage differentiation and polarization

単球由来マクロファージは自然免疫系の重要な細胞型を表す。マクロファージ生物学を勉強して、マウスのモデルは、マウスおよびヒト単球由来マクロファージの間で表現型および機能の違いに苦しんでいます。したがって、我々はここでin vitroモデル初代ヒトマクロファージを生成して勉強するについて説明します。簡単に言えば、前腕静脈から引き出さ末梢血の密度勾配遠心分離後、単球は、負磁気ビーズ分離を使用して末梢血単核細胞から単離される。これらの単球は、その後、マクロファージの分化又は異なる種類の偏光を誘導するように特定の条件の下で6日間培養する。モデルは使いやすく、マウスと人間の間に種特異的な違いに起因する問題を回避。また、不死化細胞株の使用よりもインビボ条件下に近い。結論として、ここで説明するモデルはmacrophag勉強するのに適している電子生物学は、病気のメカニズムと新規治療標的を識別します。にもかかわらず、完全に死後得動物やヒト組織を用いた実験を交換しない、ここで説明するモデルは、病気のメカニズムや様々なヒト疾患との関連性が高いかもしれない治療標的の同定と検証を可能にします。

上記のプロトコルを使用して、我々は日常的に25.1×10 6±2.2×10 6 monocytes/100 mlの血液を（26の独立した実験の平均値±標準誤差、 図1A）を取得。 CD14のためにフローサイトメトリー染色によって決定された単球純度は日常的に、95％以上（97.1±0.4％、3つの独立した実験、 図1Bからの平均±標準誤差）である。トリパンブルー染色により決定される新たに単離した単...

Watch this Scientific Journal Video about An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization at JoVE.com

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

単球由来のマクロファージは自然免疫系の重要な細胞である。ここでは、使用して簡単に説明する<em&gt; in vitroで</emこれらの細胞を生成するために&gt;モデル。勾配遠心分離、負ビーズ単離および特異的な細胞培養条件を使用して、単球由来マクロファージは、表現型および機能的研究のために生成することができる。

不定冠詞<em&gt;インビトロ</emヒトマクロファージの分化と偏光の不均一性を研究するために&gt;モデル

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

an-vitro-model-to-study-heterogeneity-human-macrophage

Research

JoVE Journal

Immunology and Infection

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

18.6K ビュー.  University of Heidelberg. 単球由来のマクロファージは自然免疫系の重要な細胞である。ここでは、使用して簡単に説明する in vitroでモデル。勾配遠心分離、負ビーズ単離および特異的な細胞培養条件を使用して、単球由来マクロファージは、表現型および機能的研究のために生成することができる。

ビデオ: An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

不定冠詞<em&gt;のin vitro</em研究する&gt;共同感染モデル<em&gt;熱帯熱マラリア原虫（Plasmodium falciparum）</emヒトの第一次単球由来の免疫細胞に&gt;-HIV-1の相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

免疫・感染学

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

骨髄由来マクロファージを用いたマクロファージ分極の調査

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

設立<em&gt;インビトロ</em&gt;システムの細胞内挙動を研究するために、<em&gt;カンジダ·グラブラタ</em&gt;人間のTHP-1マクロファージにおける

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

in vitroおよびin vivoモデルでは血管壁の下でフローの状況に細菌付着を研究するために、

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

アン<em&gt;インビトロ</em&gt; HIV重感染のコンテキストでのマラリアに対する免疫応答を測定するためのモデル

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

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 ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

マクロファージの融合を研究するための光学的品質ガラス表面を加工

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

体外分化マウス顆粒球マクロファージ ・ コロニー刺激因子 (GM-CSF)-(THGM) ヘルパー T 細胞の生産

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis and occlusive symptoms of headaches, stroke or vision loss. Macrophages and T-helper lymphocytes infiltrate the vascular wall and produce a pro-inflammatory response that lead to vessel damage and ischemia. To date, there is no GCA biomarker that can monitor disease activity and guide therapeutic response.
Folate receptor beta (FRB) is a glycosylphosphatidylinositol protein that is anchored on cell membranes and normally expressed in the myelomonocytic lineage and in the majority of myeloid leukemia cells as well as in tumor and rheumatoid synovial macrophages, where its expression correlates with disease severity. The ability of FRB to bind folate compounds, folic acid-conjugates and antifolate drugs has made it a druggable target in cancer and inflammatory disease research. This report describes the histopathologic and immunohistochemical methods used to assess expression and distribution of FRB in relation to GCAimmunopathology.
Formalin-fixed and paraffin-embedded temporal artery biopsies from GCA and normal controls were stained with Hematoxylin and Eosin to review tissue histology and identify pathognomonic features.Immunohistochemistry was used to detect FRB, CD68 and CD3 expression. A microscopic analysis was performed to quantify the number of positively stained cells on 10 selected high-power-field sections and their respective locations in the arterial wall.
Lymphohistiocytic (LH) inflammation accompanied by intimal hyperplasia and disrupted elastic lamina was seen in GCA with none found in controls. The LH infiltrate was composed of approximately 60% lymphocytes and 40% macrophages. FRB expression was restricted to macrophages, comprising 31% of the total CD68+ macrophage population and localized to the media and adventitia. No FRB was seen in controls.
This protocol demonstrated a distinct numerical and spatial pattern of the FRB macrophage relative to the vascular immune microenvironment in GCA.

The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis.

葉酸受容体ベータ マクロファージと巨細胞性動脈炎で微小血管免疫環境をプロファイリングする Immunohistopathologic 研究

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis ...