The Department of Transfusion Medicine, Clinical Center, National Institutes of Health, provided elutriated monocytes. This work was supported by the Intramural Research Program, National Heart, Lung, and Blood Institute, National Institutes of Health.

白血球搬出は、保健機関審査委員会の国立研究所によって承認されたヒト被験体の研究プロトコルの下で行いました。 1.単離と単球の凍結保存<ol><li>人間のドナーの白血球搬出によって単核細胞を得る、および参考文献7,8に記載されているように、単核細胞の連続向流遠心水簸によって単球を豊かにします。製造業者が指定する水簸バッファに- （90％単球約80）約100×10 6簸細胞を得ます。血球計数器を用いて細胞を数えます。 </li><li> 300×gで15ミリリットルポリプロピレンチューブで遠心分離した単球 室温で5分間。 </li><li>上清を除去し、穏やかに90％FBS / 10％ジメチルスルホキシド、50×10 6細胞/ mlの最終濃度を達成するようにジメチルスルホキシド、続いFBSで細胞を再懸濁します。 </li><li>細胞懸濁液tの各ミリリットルを加えます個々のクライオバイアルO。 </li><li>セル冷凍コンテナ内のクライオバイアルを置き、に転送-80℃のフリーザー24時間長期保存のために液体窒素クライオバイアル貯蔵タンクに移す前に。 </li></ol>単球からマクロファージへの2分化<ol><li>すぐに37℃の水浴に移し、細胞懸濁液は、約70％を解凍したときにすぐに除去することによって細胞のクライ​​オバイアルを解凍します。 </li><li>すぐに室温で完全に融解時、37の50ミリリットルに1ミリリットルをクライオバイアル内容を転送°Cは、2 mMのL-グルタミン、50 ngの/ mlのM-CSF、25 ngの/ mlのロズウェルパーク記念研究所（RPMI）1640培地を温めインターロイキン10（IL-10）、及び10％FBS（完全培地）。 </li><li>移送2 75cm 2のプラスチック製の細胞培養フラスコのそれぞれへの単球の懸濁液を25ml。 </li><li> 48時間、5％CO 2/95％空気で37℃で細胞培養インキュベーター中で培養物をインキュベートします。 </li><li>立方をすすぎますRPMI 1640培地を穏やかに任意の緩く付着した細胞を取り除く回避するために、培養培地を除去し、（37℃に予め温めておいた）mlの10でltures 3回。 </li><li>単球が分化し、コンフルエントになるまで十分に増殖するまで2日毎に培地を交換し、新鮮な完全培地を追加し、すすぎに続いて。これは文化の約1週間が必要です。 </li></ol> 3.収穫マクロファージ実験を開始します<ol><li> 10mlのフラスコ中で3回分化マクロファージリンス予め温めた37℃の0.25％トリプシン-エチレンジアミン四酢酸（EDTA）溶液10 mlで添加する前に、Ca 2+及びMg 2+ を含まない37℃のダルベッコのリン酸緩衝生理食塩水を予熱しました。 </li><li>マクロファージの約90％は四捨五入と切り離されている必要があり、その時点で15分 - 10のための細胞培養インキュベーターにフラスコを置きます。フラスコの顕微鏡検査することによってこれを確認してください。 </li><li> 10％FBSを含むRMPI 1640培地10 mlのSへの追加トップトリプシン処理。 </li><li> 5分間の遠心分離器、50ミリリットルのポリプロピレンチューブにフラスコから300×gでのマクロファージ細胞懸濁液を移し、上清を捨てます。 </li><li>静かに完全培地の1ミリリットル中にマクロファージを再懸濁します。 </li><li> 10μlのトリパンブルー溶液を用いてマクロファージ細胞懸濁液10μlを混合し、血球計数器を使用して、マクロファージ細胞数および生存率（典型的には&gt; 95％）を決定します。 </li><li>細胞数に基づいて、（通常は約15から18×10 5マクロファージ）、目的の播種細胞密度を達成するために追加の完全培地を追加します。注：12ウェルプレートのウェル当たり1.5ミリリットル培地中で1×10 5マクロファージを、近コンフルエント文化を生成します。 </li><li>播種後、細胞の付着を可能にするために、5％CO 2/95％空気で37℃で細胞培養インキュベーターで一晩マクロファージインキュベートします。次に、目的の実験を開始します。 </li></ol>

<ol>
	<li>Dey, A., Allen, J., Hankey-Giblin, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+polarization+of+macrophages+in+inflammation:+blood+monocytes+versus+tissue+macrophages.">Ontogeny and polarization of macrophages in inflammation: blood monocytes versus tissue macrophages.</a> Front. Immunol. 5, 683 (2014).</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>Akagawa, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+colony-stimulating+factor-induced+human+monocyte-derived+macrophages.">Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages.</a> Int. J. Hematol. 76, 27-34 (2002).</li><li>Akagawa, K. 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=Functional+heterogeneity+of+colony-stimulating+factor-induced+human+monocyte-derived+macrophages.">Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages.</a> Respirology. 11 Suppl, S32-S36 (2006).</li><li>Fleetwood, A. J., Lawrence, T., Hamilton, J. A., Cook, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+(CSF)+and+macrophage+CSF-dependent+macrophage+phenotypes+display+differences+in+cytokine+profiles+and+transcription+factor+activities:+implications+for+CSF+blockade+in+inflammation.">Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation.</a> J. Immunol. 178, 5245-5252 (2007).</li><li>Lacey, D. C., 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=Defining+GM-CSF-+and+macrophage-CSF-dependent+macrophage+responses+by+in+vitro+models.">Defining GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro models.</a> J. Immunol. 188, 5752-5765 (2012).</li><li>Strasser, E. F., Eckstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+leukocyte+collection+and+monocyte+isolation+for+dendritic+cell+culture.">Optimization of leukocyte collection and monocyte isolation for dendritic cell culture.</a> Transfus. Med. Rev. 24, 130-139 (2010).</li><li>Kim, 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=Monocyte+enrichment+from+leukapheresis+products+by+using+the+Elutra+cell+separator.">Monocyte enrichment from leukapheresis products by using the Elutra cell separator.</a> Transfusion (Paris). 47, 2290-2296 (2007).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Curr. Protoc. Immunol. Appendix 3 (Appendix 3B), (2001).</li><li>Anzinger, 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=Native+low-density+lipoprotein+uptake+by+macrophage+colony-stimulating+factor-differentiated+human+macrophages+is+mediated+by+macropinocytosis+and+micropinocytosis.">Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis.</a> Arterioscler. Thromb. Vasc. Biol. 30, 2022-2031 (2010).</li><li>Zhao, B., 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=Constitutive+receptor-independent+low+density+lipoprotein+uptake+and+cholesterol+accumulation+by+macrophages+differentiated+from+human+monocytes+with+macrophage-colony-stimulating+factor+(M-CSF).">Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF).</a> J. Biol. Chem. 281, 15757-15762 (2006).</li><li>Freeman, S. R., 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=ABCG1-mediated+generation+of+extracellular+cholesterol+microdomains.">ABCG1-mediated generation of extracellular cholesterol microdomains.</a> J. Lipid Res. 55, 115-127 (2014).</li><li>Kruth, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-independent+fluid-phase+pinocytosis+mechanisms+for+induction+of+foam+cell+formation+with+native+low-density+lipoprotein+particles.">Receptor-independent fluid-phase pinocytosis mechanisms for induction of foam cell formation with native low-density lipoprotein particles.</a> Curr. Opin. Lipidol. 22, 386-393 (2011).</li><li>Jin, X., 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=ABCA1+contributes+to+macrophage+deposition+of+extracellular+cholesterol.">ABCA1 contributes to macrophage deposition of extracellular cholesterol.</a> J. Lipid Res. 56, 1720-1726 (2015).</li><li>Ong, D. 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=Extracellular+cholesterol-rich+microdomains+generated+by+human+macrophages+and+their+potential+function+in+reverse+cholesterol+transport.">Extracellular cholesterol-rich microdomains generated by human macrophages and their potential function in reverse cholesterol transport.</a> J. Lipid Res. 51, 2303-2313 (2010).</li><li>Hashimoto, S., Yamada, M., Motoyoshi, K., Akagawa, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+macrophage+colony-stimulating+factor-induced+growth+and+differentiation+of+human+monocytes+by+interleukin-10.">Enhancement of macrophage colony-stimulating factor-induced growth and differentiation of human monocytes by interleukin-10.</a> Blood. 89, 315-321 (1997).</li><li>Hashimoto, S. I., Komuro, I., Yamada, M., Akagawa, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-10+inhibits+granulocyte-macrophage+colony-stimulating+factor-dependent+human+monocyte+survival+at+the+early+stage+of+the+culture+and+inhibits+the+generation+of+macrophages.">IL-10 inhibits granulocyte-macrophage colony-stimulating factor-dependent human monocyte survival at the early stage of the culture and inhibits the generation of macrophages.</a> J. Immunol. 167, 3619-3625 (2001).</li><li>Lund, P. K., Joo, G. B., Westvik, A. B., Ovstebo, R., Kierulf, P. <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+monocytes+from+whole+blood+by+density+gradient+centrifugation+and+counter-current+elutriation+followed+by+cryopreservation:+six+years'+experience.">Isolation of monocytes from whole blood by density gradient centrifugation and counter-current elutriation followed by cryopreservation: six years' experience.</a> Scand. J. Clin. Lab. Invest. 60, 357-365 (2000).</li><li>Weiner, R. S., Normann, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+integrity+of+cryopreserved+human+monocytes.">Functional integrity of cryopreserved human monocytes.</a> J. Natl. Cancer Inst. 66, 255-260 (1981).</li><li>Hansen, J. B., 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=Retention+of+phagocytic+functions+in+cryopreserved+human+monocytes.">Retention of phagocytic functions in cryopreserved human monocytes.</a> J. Leukoc. Biol. 57, 235-241 (1995).</li><li>Seager Danciger, 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=Method+for+large+scale+isolation,+culture+and+cryopreservation+of+human+monocytes+suitable+for+chemotaxis,+cellular+adhesion+assays,+macrophage+and+dendritic+cell+differentiation.">Method for large scale isolation, culture and cryopreservation of human monocytes suitable for chemotaxis, cellular adhesion assays, macrophage and dendritic cell differentiation.</a> J. Immunol. Methods. 288, 123-134 (2004).</li><li>Jin, X., Xu, Q., Champion, K., Kruth, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin+contamination+of+apolipoprotein+A-I:+effect+on+macrophage+proliferation--a+cautionary+tale.">Endotoxin contamination of apolipoprotein A-I: effect on macrophage proliferation--a cautionary tale.</a> Atherosclerosis. 240, 121-124 (2015).</li></ol>

The authors have nothing to disclose.

定義されたマクロファージの種類を生成することは、マクロファージの生物学を研究する研究者によって得られた相反する結果のいくつかを明確にすることができます。初代ヒトマクロファージを生成するために、さまざまな培養条件および分化因子の使用は、非常に異なるマクロファージタイプに研究者によって理解されなくてもよいことを導くことができます。例えば、マクロファージ?...

Study of cultured macrophages is a useful model to understand the function of these cells in inflammation such as occurs in atherosclerotic plaques. When the research focus is on human diseases involving macrophages, it is useful to study primary human macrophages rather than non-human macrophages to avoid species differences. Also, the effects of cell transformation can be avoided by using primary macrophages rather than macrophage cell lines. For this purpose, macrophages differentiated from monocytes isolated from human blood serve as a means of obtaining primary human macrophages. 
Tissue macrophages may be either resident within tissues or may be derived from monocytes that migrate into the tissue and differentiate into macrophages 1. Two types of human monocyte-derived macrophages have been defined that differ not only in morphology but also gene expression and cell function 2. These two types are obtained from monocytes that are differentiated into macrophages in the presence of M-CSF + fetal bovine serum (FBS) and monocytes that are differentiated into macrophages in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) + FBS 2-6. In our experience (unpublished observation), use of human serum rather than FBS generates the GM-CSF type regardless of whether M-CSF or GM-CSF is included in the differentiation medium. M-CSF type macrophages tend to be more elongated than GM-CSF type macrophages, which resemble fried eggs in their morphology. Investigators should recognize that human M-CSF and GM-CSF monocyte-derived macrophages are not the same as so called M1 and M2 mouse bone marrow-derived macrophages 6.
During research using human monocyte-derived M-CSF differentiated macrophages, we experienced difficulty related to the availability of monocytes to initiate experiments and variation in the obtained cell densities of macrophages during differentiation of monocytes into macrophages. To overcome these problems, we have developed the following protocol in which monocytes are frozen until required for use, and monocytes are differentiated in bulk into macrophages that can then be removed from culture flasks and plated at desired cell densities to obtain more uniform cultures from experiment to experiment.

<table border="0" cellpadding="0" cellspacing="0" width="1086">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Cellbind 12-well culture plate</td>
			<td style="width:319px;">Corning</td>
			<td style="width:173px;">3336</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">CELLSTAR, T-75 flask, tissue culture treated</td>
			<td style="width:319px;">Greiner Bio-One North America</td>
			<td style="width:173px;">658157</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">RPMI 1640 culture medium</td>
			<td style="width:319px;">Cellgro Mediatech</td>
			<td style="width:173px;">15-040-CM</td>
			<td style="width:167px;">warmed to 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">L-Glutamine</td>
			<td style="width:319px;">Cellgro Mediatech</td>
			<td style="width:173px;">25-005-CI</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Fetal bovine serum</td>
			<td style="width:319px;">Gibco</td>
			<td style="width:173px;">16000-036</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">M-CSF</td>
			<td style="width:319px;">PeproTech</td>
			<td style="width:173px;">300-25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">GM-CSF</td>
			<td style="width:319px;">PeproTech</td>
			<td style="width:173px;">300-03</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">IL-10&#160;</td>
			<td style="width:319px;">PeproTech</td>
			<td style="width:173px;">200-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">DMSO</td>
			<td style="width:319px;">Sigma</td>
			<td style="width:173px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Cryovial&#160;</td>
			<td style="width:319px;">Thermo Scientific&#160;</td>
			<td style="width:173px;">375418</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:428px;">DPBS without Ca2+ and Mg2+&#160;</td>
			<td style="width:319px;">Corning cellgro</td>
			<td style="width:173px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">0.25% Trypsin-EDTA&#160;</td>
			<td style="width:319px;">Gibco</td>
			<td style="width:173px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">50 ml polypropylene conical tube</td>
			<td style="width:319px;">Falcon</td>
			<td style="width:173px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Trypan Blue</td>
			<td style="width:319px;">Lonza</td>
			<td style="width:173px;">17-942E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Neubauer-improved bright light hemocytometer&#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;</td>
			<td style="width:319px;">Paul Marienfeld GmbH &#38; Co. KG</td>
			<td style="width:173px;">610031</td>
			<td>http://www.marienfeld-superior.com/index.php/counting-chambers/articles/counting-chambers.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">CoolCell LX cell freezing container</td>
			<td style="width:319px;">BioCision</td>
			<td style="width:173px;">BCS-405</td>
			<td>other freezing containers also should&#160; be adequate for this step</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Liquid Nitrogen Storage System, CryoPlus 1</td>
			<td style="width:319px;">Thermo Scientific&#160;</td>
			<td style="width:173px;">7400</td>
			<td>any liquid nitrogen tank should be adequate</td>
		</tr>
	</tbody>
</table>

culture of macrophage colony-stimulating factor differentiated human monocyte-derived macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

トリパンブルー染色9で決定されるような新鮮または凍結保存した単球の生存率は95％以上であった。 図 1および図2は 、それぞれ、より低いおよびより高い倍率でマクロファージへ新鮮で凍結保存した（ すなわち 、冷凍）単球分化の進行状況を比較します。凍結保存した単球と比較して、新鮮なが広がっていない?...

Watch this Scientific Journal Video about Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages at JoVE.com

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

マクロファージコロニー刺激因子分化したヒト単球由来マクロファージの文化

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

culture-macrophage-colony-stimulating-factor-differentiated-human

Research

JoVE Journal

Immunology and Infection

29.0K ビュー.  National Institutes of Health. A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

ビデオ: Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

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 ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

ヒト単球由来樹状細胞におけるIL-1βを用いて活性化し、NLRP3ソーム活性の測定

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

イメージングフローサイトメトリーによるヒト単球由来樹状細胞の特徴付け：二つの単球の分離プロトコルの比較

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

全血からのヒト単球由来樹状細胞の生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

フローサイトメトリーによる中枢神経系からのミクログリアおよび単球由来マクロファージの解析

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

離とマウスとヒトの肺胞マクロファージの体外培養

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

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 ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

生体内における単球血球活性の定量化と血液単球由来マクロファージの特性化

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

分化ヒト単球由来マクロファージにおける細胞外トラップ放出のIn Vitro刺激と可視化

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...