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.

白细胞分离是根据人类主体的卫生机构审查委员会的国家机构认可的研究方案进行。 1.分离的单核细胞冷冻保存<ol><li>得到单核细胞由人类供体的白细胞分离，并通过如在参考文献7,8中所述的单核细胞的连续逆流离心淘析丰富的单核细胞。得到约100×10 6淘洗细胞（约80 - 90％的单核细胞）在由制造商指定的淘析缓冲。算使用血球细胞。 </li><li>在15ml聚丙烯管中离心的单核细胞在300 xg离心 在室温下5分钟。 </li><li>除去上清液并轻轻悬浮随后二甲基亚砜FBS中的细胞达到90％的FBS / 10％二甲亚砜的终浓度和50×10 6个细胞/ ml。 </li><li>将每个毫升细胞悬液ŤØ个别冷冻管。 </li><li>放置冷冻管在细胞冷冻容器和转移至液氮冷冻管贮槽用于长期保存前转移到-80℃冷冻24小时。 </li></ol> 2.单核细胞分化成巨噬细胞<ol><li>通过快速转移到37℃水浴中，并在细胞悬浮液解冻约70％，然后立即除去解​​冻细胞的离心管。 </li><li>后立即在室温下完全融化，转移1毫升离心管内容到50ml的37℃温热罗斯韦尔园区纪念研究所（RPMI）有2mM L-谷氨酰胺，50纳克/毫升的M-CSF，25纳克/毫升1640培养基白细胞介素-10（IL-10），和10％FBS（完全培养基）。 </li><li>转印25毫升单核细胞悬浮液到每两个75cm 2的塑料细胞培养瓶中的。 </li><li>孵育培养物中，用5％ 二氧化碳 / 95％空气的37℃的细胞培养培养箱中48小时。 </li><li>冲洗立方米ltures 3次用10ml的RPMI 1640培养基（预热至37℃），轻轻除去培养基，以避免撞出任何松散附着的细胞。 </li><li>后续漂洗，加入新鲜完全培养基，培养基改变每2天，直到单核细胞分化，增殖，足以成为融合。这需要大约一个星期的文化。 </li></ol> 3.收获巨噬细胞实验启动<ol><li>用10毫升漂洗分化的巨噬细胞在烧瓶3次预热37℃的Dulbecco磷酸盐缓冲盐水不含Ca 2+和Mg 2+加入10ml预热的37℃的0.25％胰蛋白酶-乙二胺四乙酸（EDTA）溶液之前。 </li><li>此时的巨噬细胞的约90％应具有圆形和拆卸15分钟 - 在10细胞培养孵化地方烧瓶中。由烧瓶镜检验证这一点。 </li><li>加入10 mL RMPI 1640培养基含有10％FBS送顶部胰蛋白酶。 </li><li>从烧瓶中转移巨噬细胞悬浮到50ml的聚丙烯管中，离心300×g离心5分钟，并弃去上清液。 </li><li>轻轻重悬巨噬细胞在1ml完全培养基。 </li><li>混合10微升巨噬细胞悬浮液与10微升锥虫蓝溶液，并确定使用血球巨噬细胞数量和生存力（典型地&gt; 95％）。 </li><li>根据细胞计数（通常为约15 - 18×10 5个巨噬细胞），加入以达到所需接种细胞密度额外的完全培养基。注意:在每一个12孔板的孔1.5ml介质1×10 5个巨噬细胞将产生一个近汇合培养。 </li><li>以下播种，过夜孵育的巨噬细胞中，用5％ 二氧化碳 / 95％空气的37℃的细胞培养孵化器，以允许细胞附着。然后，开始所需的实验。 </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.

定义生成巨噬细胞类型可以澄清一些研究巨噬细胞生物学当调查员获得的结果相互矛盾的。使用各种培养条件和分化因子，以产生初级人类巨噬细胞可以导致非常不同的巨噬细胞的类型，可能无法由研究人员可以理解一个事实。例如，巨噬细胞有时从人单核细胞用无血清，单独的人血清，人血清补充有M-CSF，FBS单独，或含M-CSF或GM-CSF的FBS的生成。如先前报道和如我们还观察到，单核细胞为一个星期培养无血清或FBS的无添加分化因子不保持存活3。如上所述，单核细胞与人血清培养单独或补充有M-CSF产生的巨噬细胞，显示与FBS加GM-CSF分化的单核细胞的"煎鸡蛋"的形态特征。因此，在人血清的M-的存在即使单核细胞已分化与M-CSF不能得到表示多个细长形态CSF的类型的巨噬细胞。 有趣的是，我们已观察到，用含有M-CSF和GM-CSF，同时产生M-CSF细长和GM-CSF在相同的培养"煎鸡蛋"形态学表型的FBS单核细胞的分化。这表明不同的单核细胞亚群产生这两种巨噬细胞类型。以前的研究已经表明，M-CSF和GM-CSF分化因子不仅影响巨噬细胞的形态，而且也影响基因表达和细胞的功能，这是这两个细胞巨噬细胞类型2-6基本上不同。免疫染色在与由受GM-CSF的巨噬细胞表达M-CSF型巨噬细胞和25F9表达CD14结合巨噬细胞标记物CD68可用于确认巨噬细胞类型如先前2所示。这两个巨噬细胞的种类数mAY是动脉粥样硬化，其中不同类型的巨噬细胞在发生粥样硬化斑块，并能显示胆固醇代谢和炎症介质2,12,13的表达差异研究尤为重要。例如，当巨噬细胞与胆固醇12,14,15富含M-CSF型巨噬细胞是在这些巨噬细胞存胆固醇到细胞外基质独特。 中提出，与M-CSF培养过程中协议，IL-10已被常规地如先前16描述的加入。该细胞因子促进巨噬细胞的生长和分化，并产生的巨噬细胞的更均匀的细长的形态。此外，IL-10通过抑制由GM-CSF 17感应的信号事件抑制GM-CSF依赖性单核细胞的存活。然而，调查人员应该考虑到IL-10可能会影响正在研究其他细胞的反应，因此，不妨忽略IL-10。之前使用采用扩散和在一烧瓶中的单核细胞的分化当前协议，巨噬细胞的培养物通过直接电镀淘析的单核细胞进入培养孔和区分它们之前发起实验7天启动。然而，即使在加入的IL-10的，其中一些巨噬细胞培养物的结果显示"煎鸡蛋"GM-CSF型巨噬细胞的少量（通常小于10％）的污染细长M-CSF型巨噬细胞。随着当前协议，GM-CSF型巨噬细胞的污染不会对不明确的原因而发生。然而，这不是因为GM-CSF细胞巨噬细胞类型的前体的单核细胞被冷冻过程中丢失，与FBS + GM-CSF的产生优良的GM-CSF型培养物（参见图4）培养所述冷冻保存的单核细胞。 所描述的协议利用单核细胞冷冻保存它们分化成巨噬细胞之前。单核细胞冻存一直上一页iously所示不影响单核细胞的功能和通常采用18-21。在这方面，我们已经发现，通过上述存款胆固醇协议生成到类似于我们已经与该直接培养成巨噬细胞的12个非冷冻保存的单核细胞先前所描述的外空间的巨噬细胞。单核细胞冷冻保存，单核细胞总是可用的启动实验，独立的时候可以从捐赠中获得的单核细胞。而它与协议继续之前冷冻保存的单核细胞不是必须的，冻存确实产生更均匀的巨噬细胞培养在所有的巨噬细胞更传播而不是两个圆形和传播因为是当新鲜的单核细胞用于启动培养的情况。我们已经成功地产生的巨噬细胞培养物从该被冷冻保存长达6个月，（测试的最长时间）的单核细胞。在另一方面，冻存MACR播种ophages成培养实验井没有产生足够的巨噬细胞培养物。 在协议中的关键步骤包括立即将单核细胞进入冷冻以下加入二甲基亚砜，立即解冻单核细胞转移至培养基中。虽然是常见的离心冷冻细胞以去除DMSO他们被接种到培养前，我们没有发现这是在单核细胞培养成功的一个必要步骤。然而，调查可能需要做，如果残留的DMSO（2微升/毫升）的浓度比在上述协议中使用更高。不冲洗的单核细胞，直到后48小时其在培养瓶中播种是重要的。否则，某些单核细胞可以根据它们在培养表面粘附的程度会丢失。另一方面，一旦单核细胞分化成巨噬细胞，增加了巨噬细胞的粘附，可能需要更长的胰蛋白酶处理以除去它们，或细胞升降机可以用来促进初始胰蛋白酶处理后释放。如果单核细胞不增殖和分化，这可能是由于单核细胞供体的一些不寻常的特点，M-CSF的活性的损失，或者由于所使用的大量的FBS。此外，当文化不发展可能值得关注的是内毒素的试剂的污染，抑制巨噬细胞增殖22代理人。 与M-CSF瓶区分单核细胞冻存他们可以批量分化成巨噬细胞。然后，巨噬细胞可被收获并在需要的细胞密度接种到培养孔用于进行实验。利用巨噬细胞的限定数量，而不是单核细胞的定义号码发起的巨噬细胞培养物的实验中，产生了巨噬细胞培养，其中所需的细胞密度可以更一致地实现。

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.

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			<td height="21" style="height:21px;width:428px;">Cellbind 12-well culture plate</td>
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		</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.

与台盼蓝染色9确定的新鲜或冷冻保存的单核细胞的存活力大于95％。 图 1和图2在较低和较高的倍率进行比较，分别新鲜和冷冻（ 即冷冻）的单核细胞分化的进展成巨噬细胞。注意，新鲜比较了冷冻保存的单核细胞显示分化的单核细胞不传播，但仍然圆形的亚群。相朗讯液泡发生在这两个条件下，单核细胞衍生的巨噬细胞。如前所述-10,11-这些液泡代表M-CSF型巨噬细胞的macropinosomes特性。 从它们从单核细胞分化的烧瓶其收获后冷冻保存的巨噬细胞不产生成功的巨噬细胞培养物（ <s仲&gt;图3）。这种文化表现出非常稀疏的细胞附着可能是由于冷冻保存后存活率差。 用于制造M-CSF型巨噬细胞的上述协议这样做而不污染的GM-CSF型巨噬细胞的存在时，培养物直接从未经其冷冻保存和分化在烧瓶M-CSF型巨噬细胞单核细胞建立的，有时会发生。然而，单核细胞冻存与FBS分化+ GM-CSF产生卓越的GM-CSF型文化（ 图4）。 <img alt="图1" src="/files/ftp_upload/54244/54244fig1.jpg" /> 图1:新鲜与冷冻保存的单核细胞比较用于产生巨噬细胞（低倍率）冻存（冷冻）和新鲜的单核细胞的相衬显微图像的M-CSF诱导分化我期间。<html> n要巨噬细胞。示出的是在第3和7天培养瓶中培养过程中的单核细胞，并在第8天，其收获和replating到12孔培养板后一天。酒吧= 100微米，适用于所有。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54244/54244fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54244/54244fig2.jpg" /> 图2:新鲜与用于产生巨噬细胞（高倍率）冷冻保存的单核细胞单核细胞的比较中培养如在图1说明中描述并在更高的放大倍数在这里显示。相透亮空泡是macropinosomes（白色箭头）。酒吧= 50微米，适用于所有。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54244/54244fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 帐篷"FO: - together.within页保留="1"&gt; <img alt="图3" src="/files/ftp_upload/54244/54244fig3.jpg" /> 图3:冷冻保存的巨噬细胞不能产生足够的培养新鲜的单核细胞接种到烧瓶中，并分化7天M-CSF如上面图1和2。然后，分化的巨噬细胞从培养瓶中并冷藏保存收获。接着，作为用于非冻存巨噬细胞和1的影响一天（8天）在协议中描述的冷冻保存的巨噬细胞接种到12孔板中。 1天培养的相差显微镜图像示出了很少的巨噬细胞的附着以下的冷冻保存（比较每天8结果非冷藏的巨噬细胞在上面的图1）。酒吧= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54244/54244fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图4:冷冻保存的单核细胞也适用于生产的GM-CSF型单核细胞衍生的巨噬细胞冻存的单核细胞（25×10 6）接种到烧瓶中和差异8天用 ​​含有50纳克/ ml的GM-CSF的10％FBS的。相衬图像显示GM-CSF型巨噬细胞的典型的"炒鸡蛋"的形态。酒吧= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54244/54244fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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.

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

29.0K 次观看.  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.

视频: 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.

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1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 ...

科研

免疫与感染

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.

使用骨髓源性巨噬细胞巨噬细胞极化的调查

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.

激活和NLRP3炎性活性的人单核细胞来源的树突状细胞用IL-1β测量

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.

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

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.

从全血人单核细胞来源的树突状细胞的生成

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.

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

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.

小鼠与人肺泡巨噬细胞的分离和体外培养

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.

体外小鼠粒细胞-巨噬细胞集落刺激因子 (gm-CSF) 的分化--生成 t 辅助器 (tHgm) 细胞

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.

单核细胞化学策略活性在Vivo的定量和血液单核细胞衍生巨噬细胞的特征

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.

分化人类单细胞衍生巨噬细胞中细胞外陷阱释放的体外刺激与可视化

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