Name: generation of large numbers of myeloid progenitors and dendritic cell precursors from murine bone marrow using a novel cell sorting strategy
Uploaded: 2018-08-10T13:30:00.000+00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy at JoVE.com

我们感谢在奥本大学兽医医学流式细胞仪设施的艾莉森教堂鸟的技术援助, 从 NIH 到 EHS R15 R15 AI107773, 以及在奥本大学的细胞和分子生物学项目的资助为夏季研究基金提供资金。

所有动物工作都是由奥本大学机构动物保育和使用委员会根据《国家卫生研究院实验室动物护理和使用指南》中概述的建议批准的。
1. 骨髓收集的准备工作
<ol><li>准备250毫升完整的媒体, 通过添加一个解决方案的罗斯威尔公园纪念研究所 (RPMI) 1640 培养基补充10% 胎小牛血清, 2 毫米谷氨酰胺, 50 µM 2 巯基乙醇到0.22 µM 真空过滤瓶单元顶部, 并应用真空。 注: 完全培养基可储存在4摄氏度, 长达2月。</li>
	<li>在2毫升瓶中混合350毫升100% 乙醇与150毫升不育 H500O, 制备70% 乙醇溶液。</li>
	<li>将离心机设置为4摄氏度。</li>
	<li>用70% 乙醇消毒镊子、手术刀和剪刀。</li>
	<li>使用血清学吸管, 添加5毫升完整的培养基到三60毫米培养皿。</li>
	<li>使用血清学吸管, 添加30毫升完整的介质到50毫升圆锥管。</li>
</ol>2. 小鼠骨髓细胞的收集
<ol><li>根据2013美国兽医协会 (AVMA) 《安乐死准则》制定的规则, 弄死一只 C57BL/6 老鼠, 共2麻醉。</li>
	<li>
取下并剥下后腿。
	<ol>
<li>用75% 乙醇将后腿和躯干饱和, 用弯曲的组织剪刀在髋部周围的皮肤上进行浅切口。使用镊子, 牢牢地将皮肤从臀部向下拉向脚踝, 露出肌肉。使用剪刀去除皮瓣。</li>
		<li>通过穿过股骨/髋关节上方的骨骼来切除整条后腿。 注: 除消毒区域外, 乙醇还有助于提供清洁的切口, 防止毛发污染样品。</li>
		<li>如果腿部将被转移到一个新的位置, 骨髓采集, 淹没的腿在完全的媒体。</li>
	</ol>
</li>
	<li>在无菌生物安全柜中工作, 将腿部转移到以前准备好的培养皿中。</li>
	<li>用剪刀剪下脚踝, 小心地取出尽可能多的肌肉和弹性结缔组织。把被清洗的骨头转移到第二个准备好的培养皿上。 注: 虽然没有必要去除所有的肌肉, 过多的剩余组织可能会使它难以冲洗出骨髓。</li>
	<li>
把股骨、膝盖和胫骨分开。
	<ol>
<li>用镊子, 把腿放在膝盖上, 找到骨髓。 注: 骨髓应可见, 在股骨顶部和胫骨末端的骨腔内有一条微弱的红线。<ol>
<li>使用剪刀, 使三削减如下。<ol>
<li>在骨髓似乎结束的地方切开胫骨。</li>
				<li>就在膝关节下面切。</li>
				<li>就在膝关节上方切开。</li>
				<li>如果髋关节仍与股骨相连, 请在髋关节下方切开。</li>
			</ol>
</li>
			<li>将三片段返回到培养皿, 并在另一条腿上重复此过程。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
从股骨和胫骨中冲洗骨髓。
	<ol>
<li>填充一个10毫升注射器与完整的媒体从50毫升圆锥管, 并帽与23克针。</li>
		<li>在第三个已准备好的培养皿上方用镊子将骨夹住, 将针头插入骨管中, 并将其推进, 冲洗出细胞 (可能出现为完整的 "插头" 或多个簇)。重复, 直到没有更多的颜色可以看到通过骨头。必要时用介质填充注射器。</li>
	</ol>
</li>
	<li>
粉碎骨骺。
	<ol>
<li>当仍在第二培养皿中, 用镊子牢牢地握住膝盖帽, 并用注射器的尖端将膝盖捣碎。继续, 直到骨骺不再是红色的。</li>
	</ol>
</li>
	<li>使用注射器, 将第二和第三培养皿中的细胞转移到50毫升管。通过轻轻吹打的向上和向下分裂成团簇。尽量不要产生气泡。离心机在 250 x g在4°c 为10分钟。</li>
	<li>
溶解红细胞
	<ol>
<li>用血清吸管去除上清液, 通过弹球和溶解红细胞, 在室温下通过1毫升的 ACK (氯化铵-氯化钾) 裂解缓冲剂孵化出1分钟。</li>
		<li>使用血清学吸管, 加入40毫升的 HBSS (汉克斯平衡盐溶液) 缓冲。</li>
	</ol>
</li>
	<li>使用血清学吸管, 过滤细胞, 虽然70µm 细胞过滤器进入一个新的50毫升锥管。离心机在 250 x g在4°c 为10分钟。</li>
	<li>使用血清吸管, 去除上清和洗涤细胞与40毫升完整的培养基。离心机在 250 x g在4°c 为10分钟。 注意: 在这个阶段, 血统阳性淋巴细胞可以通过外地或磁柱纯化去除。然而, 淋巴细胞在培养中不长期保持。虽然在骨髓 Ly6C-CD115 中有大量的谱系阳性细胞存在, 但几乎所有5天都缺席 (图 2)。</li>
	<li>使用血清吸管, 去除上清, 并培养完整的培养基中的骨髓细胞与 10 ng/毫升的重组小鼠脑脊液在密度为 1 x 106细胞/毫升。 注: 一般情况下, 红细胞裂解后可收获 4 x 107的总细胞。但是, 对于初学者和多达 5 x 107个单元的经验丰富的收割机来说, 只需要 2 x 107 。</li>
	<li>使用血清学吸管, 将细胞转移到组织培养板上, 在 5% CO2中孵育37摄氏度。如果使用24井板, 种子每井与2毫升细胞悬浮。</li>
	<li>每48小时, 使用一个血清学吸管删除一半的媒体和替代新鲜完整的媒体和转基因脑脊液。 注: 文化可以保持9天。然而, 作文随着时间的推移而改变。有关详细信息, 请参阅第3节。</li>
</ol>3. 选择日排序
<ol><li>随着时间的推移, 细胞数量的变化, 选择一个产生最高数量的期望细胞的一天。</li>
	<li>请参见表 1 , 以预测每 1 x 107个细胞在3、5和7天内培养的每一个种群的预期细胞产量。</li>
</ol>4. 染色策略
<ol><li>使用小整除数的细胞来准备控制样品。包括一个无瑕控制, 只有一个荧光抗体染色的补偿控制样本, 和荧光-减号-一个控制, 其中所有的抗体添加除了一个, 以控制在该通道的非特异荧光。如果使用间接标记, 则仅包括初级、次要和初级和次要。 注: 藻红蛋白 (PE) 和复方阿司匹林 (APC) 标记的抗体提供了独特的分离与最小出血时一起使用。但是, 如果 fluorochrome 选项是有限的, CD115 是在相对较低的水平, 而 Ly6C 是在非常高的水平表达。因此, 更明亮的荧光是可取的 anti-CD115, 并选择 anti-Ly6C 荧光, 以防止出血。</li>
	<li>在50毫升锥形管中混合97毫升的冷冻 Dulbecco 磷酸盐缓冲盐水 (DPBS) 和3毫升的胎儿小牛血清, 并将其放置在冰浴中, 准备100毫升的 FWB 洗涤缓冲液。</li>
	<li>使用吸管轻轻, 但彻底, 吸管细胞向上和向下, 以去除任何松散黏附的细胞。</li>
	<li>
使用血清吸管, 转移细胞到50毫升圆锥管。离心机在 250 x g在4°c 为10分钟。
	<ol>
<li>如果细胞体积超过50毫升, 转移细胞到必要数量的管, 并结合在染色步骤。</li>
	</ol>
</li>
	<li>轻轻地倒上清液, 用血清吸管加入30毫升的 FWB 来洗涤颗粒细胞。离心机在 250 x g在4°c 为10分钟, 并且重复洗涤。 注: 如果预期高细胞死亡, 细胞可以用 FWB 的低至0.5% 胎小牛血清 (FCS) 进行冲洗。这将防止细胞结块。</li>
	<li>
根据抗体制造商的说明暂停和染色细胞。
	<ol>
<li>悬浮 5 x 107细胞在1毫升 FWB 并且增加2µg 每个 anti-Ly6C 和 anti-CD115 标记与显影 (研究员的选择)。为了进一步区分 CMP 与 moDC (两者都是 Ly6C 和 CD115), 添加2µg anti-CD11c 抗体 (CMP 是 CD11c-; moDC 是 CD11c+)。在冰上孵化30分钟。 注:图 3是使用 Ly6C-PE 和 CD115-APC 生成的。</li>
	</ol>
</li>
	<li>使用血清学吸管, 添加10毫升的 FWB, 离心机在 250 x克4 摄氏度, 10 分钟。</li>
	<li>轻轻地倒上清液, 用血清吸管加入30毫升的 FWB 来洗涤颗粒细胞。离心机在 250 x g在4°c 为10分钟, 并且重复洗涤。</li>
	<li>在悬浮细胞之前, 要彻底地将球管取出。使用血清吸管悬浮细胞在 1 x 107细胞/毫升 FWB, 并筛选通过35µm 细胞过滤器。使用血清学吸管将过滤过的细胞转移到聚丙烯管中, 放在冰上, 直到准备好排序。 注: 如果聚丙烯不可用, 蛋白质涂层 (脱脂干牛奶或 FCS) 聚苯乙烯管可用于减少粘结。</li>
</ol>5. 根据控制样本设置闸门
注意: 为了防止由于高速流流的压力而导致细胞中断, 请使用100–130µm 喷嘴进行细胞分类。
<ol><li>
运行无瑕控件 (参见步骤 4.1) 通过单元格分拣器, 并应用一个门排除小碎片 (低正向散射;FSC) 和高度粒状 (高边分散;SSC) 粒子。
	<ol>
<li>要只分析最新的阶段 (单核细胞, moMac/MoDP 和 MoDC), 应用门控仅包括较大的细胞 (高 FSC)。</li>
		<li>如果使用了生存能力污点, 请使用这些污渍排除染色的、不可生存的事件 (如图 1所示)。</li>
	</ol>
</li>
	<li>通过单元分拣器运行单个荧光控制样本, 并根据需要调整补偿。</li>
	<li>运行多标签示例的示例。观察四种不同的种群: Ly6C+CD115 (GMPs)、Ly6C+CD115+ (单核细胞)、Ly6C-CD115+ (moMacs/moDP) 和 Ly6C-CD115-(中医/moDCs)。应用一个门来隔离四个主要种群。 注: 中医与 moDC 共 Ly6C-CD115 表型。但是, 它们可以根据 CD11c 表达式进行区分: CMP 缺乏 CD11c, 而 MoDCs 表达 CD11c。</li>
</ol>6. 孤立人口的收集
<ol><li>通过添加足够的 FCS, 以达到至少20% 最终浓度时, 充分的准备收集管。例如, 如果使用5毫升管, 在分拣前添加1毫升的 FCS, 并在达到5毫升总容积时取出管。</li>
	<li>
为了防止膜的更替和抗体摄取, 保持所有样品 (混合和排序) 在4°c 在整个排序。
	<ol>
<li>如果这是不可能的, 保留包含细胞的管子在冰尽可能被排序并且转移整除数到分拣机根据需要。</li>
		<li>另外, 将排序的样品转移到冰每20–30分钟。</li>
	</ol>
</li>
	<li>在收集了所需的细胞数之后, 使用血清学吸管将细胞转移到新的锥形管上。离心机在 250 x g在4°c 为10分钟。<ol>
<li>确认纯度与后排序分析的小整除数从每个收集的人口。</li>
	</ol>
</li>
	<li>除去上清, 在10毫升的 FWB 和离心机悬浮在 250 x g在4°c 为10分钟. 重复两次洗涤。 注: 去除所有上清液可能很难, 不逐出颗粒。将吸管尖端连接到真空线可以帮助去除。如果没有这种情况, 建议将上清液在新鲜的试管中收集, 以防颗粒分离。</li>
	<li>第二次冲洗后取出上清液。</li>
	<li>如果用户的实验设计要求重新培养细胞, 请按照步骤2.12–2.14。否则, 如果单元格用于立即分析, 则根据所需的协议准备单元格。 注: 典型功能分析示例包括在图 4中。</li>
</ol>

<ol>
	<li>Zhan, Y., Xu, Y., Lew, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+the+development+and+function+of+dendritic+cell+subsets+by+GM-CSF:+more+than+a+hematopoietic+growth+factor.">The regulation of the development and function of dendritic cell subsets by GM-CSF: more than a hematopoietic growth factor.</a> Mol Immunol. 52, 30-37 (2012).</li><li>Zhan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+cytokine,+GM-CSF,+alters+the+developmental+outcome+of+murine+dendritic+cells.">The inflammatory cytokine, GM-CSF, alters the developmental outcome of murine dendritic cells.</a> Eur J Immunol. , (2012).</li><li>van de Laar, L., Coffer, P. J., Woltman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+dendritic+cell+development+by+GM-CSF:+molecular+control+and+implications+for+immune+homeostasis+and+therapy.">Regulation of dendritic cell development by GM-CSF: molecular control and implications for immune homeostasis and therapy.</a> Blood. 119, 3383-3393 (2012).</li><li>Steinman, R. M., Inaba, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+dendritic+cells.">Myeloid dendritic cells.</a> J Leukoc Biol. 66, 205-208 (1999).</li><li>Inaba, K., 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=Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte/macrophage+colony-stimulating+factor.">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> J Exp Med. 176, 1693-1702 (1992).</li><li>Steinman, R. M., Pack, M., Inaba, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+development+and+maturation.">Dendritic cell development and maturation.</a> Adv Exp Med Biol. , 1-6 (1997).</li><li>Pierre, 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=Developmental+regulation+of+MHC+class+II+transport+in+mouse+dendritic+cells.">Developmental regulation of MHC class II transport in mouse dendritic cells.</a> Nature. 388, 787-792 (1997).</li><li>Steinman, R. M., Witmer-Pack, M., Inaba, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells:+antigen+presentation,+accessory+function+and+clinical+relevance.">Dendritic cells: antigen presentation, accessory function and clinical relevance.</a> Adv Exp Med Biol. , 1-9 (1993).</li><li>Na, Y. R., Jung, D., Gu, G. J., Seok, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+Grown+Bone+Marrow+Derived+Cells+Are+Composed+of+Phenotypically+Different+Dendritic+Cells+and+Macrophages.">GM-CSF Grown Bone Marrow Derived Cells Are Composed of Phenotypically Different Dendritic Cells and Macrophages.</a> Mol Cells. 39, 734-741 (2016).</li><li>Rogers, P. B., Driessnack, M. G., Hiltbold Schwartz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+developmental+stages,+kinetics,+and+phenotypes+exhibited+by+myeloid+cells+driven+by+GM-CSF+in+vitro.">Analysis of the developmental stages, kinetics, and phenotypes exhibited by myeloid cells driven by GM-CSF in vitro.</a> PLoS One. 12, e0181985 (2017).</li><li>Helft, 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=GM-CSF+Mouse+Bone+Marrow+Cultures+Comprise+a+Heterogeneous+Population+of+CD11c(+)MHCII(+)+Macrophages+and+Dendritic+Cells.">GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c(+)MHCII(+) Macrophages and Dendritic Cells.</a> Immunity. 42, 1197-1211 (2015).</li><li>Alloatti, A., Kotsias, F., Magalhaes, J. G., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+maturation+and+cross-presentation:+timing+matters!.">Dendritic cell maturation and cross-presentation: timing matters!.</a> Immunol Rev. 272, 97-108 (2016).</li><li>Jakubzick, C. V., Randolph, G. J., Henson, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+differentiation+and+antigen-presenting+functions.">Monocyte differentiation and antigen-presenting functions.</a> Nat Rev Immunol. 17, 349-362 (2017).</li><li>Mbongue, J. C., Nieves, H. A., Torrez, T. W., Langridge, W. 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+Role+of+Dendritic+Cell+Maturation+in+the+Induction+of+Insulin-Dependent+Diabetes+Mellitus.">The Role of Dendritic Cell Maturation in the Induction of Insulin-Dependent Diabetes Mellitus.</a> Frontiers in immunology. 8, 327 (2017).</li><li>Shortman, K., Naik, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steady-state+and+inflammatory+dendritic-cell+development.">Steady-state and inflammatory dendritic-cell development.</a> Nat Rev Immunol. 7, 19-30 (2007).</li><li>Xu, Y., Zhan, Y., Lew, A. M., Naik, S. H., Kershaw, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+development+of+murine+dendritic+cells+by+GM-CSF+versus+Flt3+ligand+has+implications+for+inflammation+and+trafficking.">Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking.</a> J Immunol. 179, 7577-7584 (2007).</li><li>Naik, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+large+numbers+of+pro-DCs+and+pre-DCs+in+vitro.">Generation of large numbers of pro-DCs and pre-DCs in vitro.</a> Methods Mol Biol. 595, 177-186 (2010).</li><li>Nikolic, T., de Bruijn, M. F., Lutz, M. B., Leenen, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+stages+of+myeloid+dendritic+cells+in+mouse+bone+marrow.">Developmental stages of myeloid dendritic cells in mouse bone marrow.</a> Int Immunol. 15, 515-524 (2003).</li><li>Hettinger, 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=Origin+of+monocytes+and+macrophages+in+a+committed+progenitor.">Origin of monocytes and macrophages in a committed progenitor.</a> Nat Immunol. 14, 821-830 (2013).</li><li>Fogg, D. K., 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=A+clonogenic+bone+marrow+progenitor+specific+for+macrophages+and+dendritic+cells.">A clonogenic bone marrow progenitor specific for macrophages and dendritic cells.</a> Science. 311, 83-87 (2006).</li><li>Traver, D., 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=Development+of+CD8{alpha}-Positive+Dendritic+Cells+from+a+Common+Myeloid+Progenitor.">Development of CD8{alpha}-Positive Dendritic Cells from a Common Myeloid Progenitor.</a> Science. 290, 2152-2154 (2000).</li></ol>

作者没有冲突要公开。

该协议促进了基因型脑脊液驱动的祖细胞和前体细胞类型的分离, 这些数字足以进行多种类型的分析, 包括生化化验、体外细胞功能的测定或体内灌注。这种方法代表单核细胞衍生的树突状细胞的发展, 使细胞在这一发展途径的早期可靠的分离和鉴定, 以及那些分化的细胞类型更常见的一个重要进展在先前的研究中被孤立。
以前的分离从骨髓中产生的祖细胞和前体的协议依赖于 CFSE 染色, 以确定增生祖体细胞17或染色与 CD31 和 Ly6C 作为髓细胞的标志物18.奈克描述的 CFSE 染色协议设计用于 Flt3L-driven DC 祖细胞和前体17的生成。当我们在 GM-CSF 文化系统中尝试这种方法时, 我们遇到了两个主要问题。CFSE 对发育中的细胞有一定的毒性, 而且是如此的明亮 (即使是在分裂的细胞中), 它使补偿变得困难。这种方法被证明不适合我们的目标, 孤立早期细胞类型 (祖), 以及细胞跨越发展光谱, 从高度增生 (中医/GMPs) 高度发展 (MoDC/MoMac)。我们还尝试了基于 CD31 和 Ly6C 18的 Leenen 组描述的 GM-脑脊液驱动细胞的分类策略。然而, 我们发现 CD31 是有问题的。它被表达在非常低的水平 (使它难以解决人口的清洁排序) 由一个微乎其微的小子集的细胞, 并且仅非常早期在文化期间。事实上, CD31 的表达在 GM-CSF 10的2天的文化中是无法检测到的。
Ly6C, 然而, 是一个非常有用的分子分离细胞在不同的发展阶段, 由于其瞬时模式的表达10。添加 CD115 使我们能够更密切地辨别细胞在发展的中期和后期阶段, 这是不可能与 CD31。我们还研究了其他祖先的标记物, 如 CD34 和 CD117, 希望将大量这些早期细胞隔离10。然而, 与在 Flt3L 系统中报告的不同, 我们发现 CD34 和 CD117 也被表达在一个非常小的细胞子集上, 几乎没有在3天的文化17。然而, 这些干细胞标记在未来的研究中可能是有用的, 以区分 CMP 或 GMP 人群中的子集。
对可能影响所需细胞种群产量的议定书有几种可能的修改。首先, 用户可以选择应用生存性污点排除任何死细胞。根据观察, 典型的前、侧散射门内的死细胞率一般较低, 仅在最初几天的培养过程中出现问题, 与谱系阳性淋巴细胞的减少相吻合。然而, 对于细胞数必须精确的应用, 生存的污点将确保排除死细胞。
第二种可能的改变是在培养之前, 血统阳性淋巴细胞的损耗。我们已经尝试这种方法来解决在 Ly6C-CD115 细胞群中经常观察到的谱系阳性细胞的小种群。总细胞产量在5和6天略有降低, 纯度不显著高于 (未显示数据)。因此, 纯度没有理由降低产量。同样, 如果用户计划在5天或之后进行排序, 则文化中淋巴细胞的频率远远低于 1%, 不应出现问题。
最后, 因为这个策略是为了隔离一个大的发展频谱的细胞, 用户应该调整排序的时间, 以收集尽可能多的预期人口。此排序策略忠实地生成所指示的单元格类型, 而不管单元格排序的区域性是哪一天。例如, 具有表型 Ly6C+ CD115-CD11c 的细胞是真实的 GMP 表型和功能相似, 无论是排序和隔离在3天或5的文化。然而, 这些细胞的频率在3天比5日大得多, 所以如果目标是隔离这种细胞类型, 建议在3天进行排序。同样值得注意的是, 在3天的进展过程中, 细胞的反应速度比在5或7天 (图 3) 的进化阶段慢。
虽然这种方法允许明确划定和隔离的5–6不同的种群沿发展光谱, 有可能更多的人口或过渡阶段沿着这一途径。在五个被描述的人口中, 可能有几个子种群的发展略有不同。例如, 我们观察到了 "截然不同" 的人口, 中间的 CD115 和 Ly6C 的发展模式略有不同, 在产生多少 moMac 和 moDC (未显示的数据) 方面。也有可能是先前在体内观察到的种群存在于培养和分类系统中, 但由于它们的频率非常低, 很难识别。cMOP 19、MDP 20或 CDP 21等种群可能存在, 但可能在较大的人群中被遮蔽。今后的研究将需要进一步澄清在这个分类框架内可能被隔离的许多发展阶段, 加上特定的差别标记。这种分类策略的价值在于, 它允许在 DC 个体发育过程中对大量发育不同阶段的连续隔离。

细胞因子-巨噬细胞集落刺激因子 (GM-CSF) 培养小鼠骨髓细胞的方法广泛应用于大数1的单核细胞衍生树突状细胞 (moDC; 也称为炎症 DC) , 2,3,4,5。这些细胞在树突状细胞 (DC) 功能6,7,8的各种研究中非常有用。通常, 这些小鼠骨髓细胞培养6-8 天, 然后用于研究树突状细胞功能5。这些文化长期被认为主要是同源的, 包括多数被区分的 moDC。最近, 很明显, 在这个6–8的天文化时期结束时, 确实有许多 moDC, 以及分化的单核细胞衍生巨噬细胞 (moMacs) 9,10,11的大子集。我们自己的研究进一步扩展了这些发现, 证明其他不发达细胞的子集, 如 moDC 前体 (moDP) 和单核, 在7天10的时间内仍然保持在低频率的文化中。因此, 利用这个系统产生的细胞来研究树突状细胞 (DC) 功能, 可以反映出比以前更广泛的细胞类型的反应。
我们从研究 GM-CSF 生成的 moDC 与这些细胞在分化的最后阶段12,13,14的功能上学到了很多。然而, 我们对这些细胞2、15、16的发展途径以及它们如何和何时表现出特定功能, 如: 对病原体相关分子的反应能力的了解明显较少。模式 (PAMPs), 吞噬功能, 抗原处理和表现13, 和抗菌活性。17报告了一项隔离大量常规 Flt3L-driven DC 祖和前体的议定书。利用羧基琥珀酰亚胺酯 (CFSE) 染色的骨髓细胞 (在 Flt3L 中跟踪分裂细胞) 和培养3天, 实现了这些不同种群的分离。细胞被耗尽的宗族阳性细胞, 并排序为祖和前体群体的基础上 CD11c 表达17。Leenen 组在 GM-脑脊液驱动培养中识别 DC 早期祖细胞的另一种方法是根据 CD31 和 Ly6C 18对单元进行分类。最初的目标是建立一个类似的方法来获得 GM-脑脊液驱动 moDC 的祖和前体。由于由 GM-CSF 产生的特定细胞类型, 我们根据在早期和后期的发展阶段所表达的分子表达方式, 调整了方法和排序策略。我们最终确定 Ly6C、CD115 (CSF-1 受体) 和 CD11c 是区分这些细胞类型10的最佳标记。
在这里, 我们提出了一个方法, 在几个不同的发展阶段的细胞分离的途径, 由 GM-脑脊液: 普通髓样祖 (CMP), 粒细胞巨噬祖 (GMP), 单核细胞源性巨噬细胞(MoMac) 和单核细胞衍生 DC (MoDC)。moMac 种群可以根据 MHC II 类表达的水平进一步隔离, 揭示了 moDC 前体种群 (moDP) 10。我们利用一种高速荧光活化细胞分选 (Ly6C) 策略来隔离这5种群, 并以表达 CD115 和 CD11c 为基础。然后, 我们展示了这些细胞在功能检测中的检查, 揭示了它们对玉兰刺激的反应。

<table><tbody><tr><td>RPMI 1640</td><td>Corning</td><td>15-040-CV</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>HyClone</td><td>SV30014.04</td><td>to supplement complete medium and FWB</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050</td><td>to supplement complete medium</td></tr><tr><td>2-mercaptoethanol (2-ME)</td><td>MP Biomedical</td><td>190242</td><td>to supplement complete medium</td></tr><tr><td>75 mM Vacuum Filter</td><td>Thermo Scientific</td><td>156-4045</td><td>to sterilize complete media</td></tr><tr><td>ACK Lysis Buffer</td><td>Lonza</td><td>10-548E</td><td>to lyse red blood cell</td></tr><tr><td>HBSS buffer</td><td>Corning</td><td>21-020-CM</td><td>to rescue leukocytes after red blood cell lysis</td></tr><tr><td>Phosphate Buffered Saline (PBS), Dulbecco&#039;s</td><td>Lonza</td><td>17-512F</td><td>must be endotoxin free; chilled at 4 &amp;deg;C</td></tr><tr><td>35 &amp;micro;m Cell filter</td><td>Falcon</td><td>352235</td><td>to break apart clumps before running through cytometer.</td></tr><tr><td>GM-CSF</td><td>Biosource</td><td>PMC2011</td><td>usable concentration of 10 ng/mL</td></tr><tr><td>Tissue cultured treated plate</td><td>VWR</td><td>10062-896</td><td>for bone marrow cells after harvest</td></tr><tr><td>Anti-Ly6C, Clone HK1.4</td><td>Biolegend</td><td>128018</td><td></td></tr><tr><td>Anti-CD115, Clone AFS98</td><td>Tonbo Bioscience</td><td>20-1152-U100</td><td></td></tr><tr><td>Anti-CD11c, Clone HL3</td><td>BD Biosciences</td><td>557400</td><td>to differeniate CMP and MoDCs</td></tr><tr><td>MoFlo XPD Flow Cytometer</td><td>Beckman Coulter</td><td>ML99030</td><td></td></tr><tr><td>BD Accuri C6</td><td>BD Biosciences</td><td>660517</td><td></td></tr><tr><td>100% Ethanol</td><td>Pharmco-Aaper</td><td>111000200CSPP</td><td></td></tr><tr><td>60 mm Petri Dish</td><td>Corning, Inc</td><td>353002</td><td></td></tr><tr><td>50 mL Conical tube</td><td>VWR</td><td>21008-242</td><td></td></tr><tr><td>C57BL/6 Mice</td><td>The Jackson Laboratory</td><td>000664</td><td>Female; 10-20 weeks old</td></tr><tr><td>Biosafety Hood</td><td>Thermo Scientific</td><td>8354-30-0011</td><td></td></tr><tr><td>10 mL Syringe</td><td>BD Biosciences</td><td>301604</td><td></td></tr><tr><td>23 G needle</td><td>BD Biosciences</td><td>305145</td><td></td></tr><tr><td>Centrifuge 5810&amp;nbsp;R</td><td>eppendorf</td><td>22625501</td><td></td></tr><tr><td>FlowJo Software v10</td><td>BD Biosciences</td><td>Version 10</td><td>flowjo.com</td></tr></tbody></table>

generation of large numbers of myeloid progenitors and dendritic cell precursors from murine bone marrow using a novel cell sorting strategy

细胞-巨噬细胞集落刺激因子 (GM-CSF) 在小鼠骨髓中产生的单核细胞来源的树突状体细胞 (moDC) 的培养最近被认为比以前所赞赏的多异质性。这些文化通常包含 moDC 和单核细胞衍生巨噬细胞 (moMac), 甚至一些较不发达的细胞, 如单核。本议定书的目标是提供一种一致的方法来识别和分离这些文化中存在的许多细胞类型, 以便它们的具体功能可以进一步研究。这里提出的分类策略将细胞先分为四种, Ly6C 和 CD115 的表达, 它们在转基因-脑脊液驱动的文化中发展的时候, 都是由细胞瞬时表达的。这四个种群包括普通髓样祖细胞或 CMP (Ly6C、CD115)、粒细胞/巨噬细胞祖或 GMP (Ly6C+、CD115)、单核细胞 (Ly6C+、CD115+) 和单核细胞衍生的巨噬菌或 moMac (Ly6C、CD115+)。CD11c 还添加到排序策略中, 以区分 Ly6C 中的两个种群, CD115-人口: CMP (CD11c) 和 moDC (CD11c+)。最后, 根据 MHC II. 类表达的水平, 在 Ly6C、CD115+ 人群中可以进一步区分两个种群。MoMacs 表达较低水平的 mhc ii 类, 而单核细胞衍生的 DC 前体 (moDP) 表达较高的 mhc ii. 类。这种方法可以可靠地隔离几个发育不同的种群, 这些数字足以用于各种功能和发展分析。我们强调一个这样的功能读数, 这些细胞类型的差异反应的刺激与病原体相关的分子模式 (PAMPs)。

为了尽可能多地进行分析, 可以根据前向和侧向散射来常规地选择可行的细胞, 不包括非常小和非常粒状的事件 (在图 1A中的所有点地块上都应用一个典型的门)。为了确定这种门控策略是否可靠地排除了死细胞, 我们染色了7氨基放线菌 D (7) (图 1B)。7AAD. 由于细胞膜通透性, 在死亡和死亡细胞中染色 DNA, 并被存活细胞排除。分析了转基因-脑脊液培养骨髓细胞的生存能力, 立即进行了收获后 (ph 值) 和1、3和 5 ph 值细胞分析, 当一个典型的 FSC/SSC 门被应用到从骨骼中分离出来的新鲜细胞时, ph 值有大约 10% 7 反正细胞。骨髓 (图 1, 收获后)。同样比例的死亡细胞也出现在1天 (~ 12%) 和3天 (~ 11%) 的文化 (图 1, 1 天 ph 值和3天 ph 值)。到了5天, 门内死细胞的数量减少到了 5% (图 1, 5 天的 PH 值)。因此, 使用这样的生存能力门通常适合在5天和之后排序。因此, 如果用户的可用参数有限, FSC/SSC 门通常是适当的。然而, 对于更敏感的化验 (特别是如果他们依赖于精确的细胞数), 纳入一个生存能力染色 (建议)。
许多树突状细胞的传播协议建议在培养11、17之前从骨髓中耗尽血统阳性细胞, 特别是淋巴细胞。这一过程被认为是提高细胞纯度的恢复后, 由 GM-脑脊液介导的分化。通常, 用磁性珠子或细胞分选11,17,18来表示 T 细胞 (CD3)、B 细胞 (CD45R 或 CD19) 和 NK 细胞 (NK1.1) 的标记物可能耗尽。然而, 根据培养系统, 淋巴细胞很少恢复或检测到这些化验。因此, 我们试图评估在 Ly6C-CD115-脑脊液驱动细胞中维持的淋巴细胞的寿命 (图 2A)。GM-脑脊液培养的骨髓细胞 (没有血统耗尽) 被染色的抗体 CD3, CD45R 和 NK1.1 (在同一荧光) 和每日测量流式细胞术 (图 2B)。在 Ly6C-CD115 人群中, CD3/CD45R 阳性细胞持续0–3天 (图 2B)。在4天, 只有少数 CD3/CD45R 阳性细胞保持, 并在5天和 6, 没有 CD3/CD45R 表达细胞存在。因此, 在 GM-CSF 的4天的文化中, 血统阳性细胞基本上是缺席的, 并没有发现在所有的天5和6的文化。
随着细胞的发育和分化 (表 1 , 基因型脑脊液驱动细胞培养的组成在这个系统中每天变化;图 3)。在早期的时间点, 最丰富的细胞是祖和前体, 并在晚些时候大多数细胞被区分10。为了确定不同文化天的排序如何影响后续的发展路径或动力学, 排序执行3、5和7天的 PH 值 (图 3A)。MoMac 人口 (Ly6C-CD115+) 的发展然后被追踪了在2–3进一步天在文化 (图 3B-3D)。
当3天的 PH 值排序时, 只有40% 的 Ly6C-CD115+ 细胞在 24 h 后排序 (PS) 中减少 CD115 表达 (图 3B)。由 48 h PS, 下调节 CD115 的分数为 66%, 72 h, 70% 的细胞有这种表型。这个表型构成被维护了 (~ 70-72% Ly6C-CD115-) 甚而在进一步天以后文化 (数据没有显示)。当5天的 PH 值, 75% 的细胞被 Ly6C-CD115-, 在24小时内迅速下调 CD115, 80% 是 CD115-仅次于48小时。这个发行在 72 h 以后被维护了 (图 3C)。最后, 当7天的 PH 值排序时, CD115 的下调节也相当迅速。在24小时内, ~ 75% 的细胞有下调 CD115 表达, 这一趋势保持在 48 h (图 3D)。有趣的是, 在7天排序时, CD115 表达的整体水平在 CD115+ 人群中的细胞中较低。
因此, 这些发现表明, 在早期排序时, 发育的动力学速度有些慢, 如3天排序的细胞相比, 在5或7天排序后观察到的更快的发展和差异。根据这些结果, 寻求更大数量 moDC 的用户在5或7天可能会排序。
DC 的成熟响应建立在6、7、12、14之间。当处理与各种病原体相关的分子模式 (PAMPs), 未成熟的 DC 上调的表达 MHC, 刺激分子和促炎性细胞因子, 提高其 T 细胞活化能力6。然而, 当发育中的细胞获得对玉兰刺激反应的能力以及它们可能呈现的 DC 成熟的特征时, 就不那么清楚了。为了确定哪些排序的人口将响应成熟刺激, 每一个人口在排序后不久, PAMPs 的鸡尾酒, 包括聚 I: C, 脂多糖 (LPS), 和 CpG DNA, 以触发类似收费受体 (TLR) 3 (TLR3),TLR4 和 TLR9 分别。用流式细胞仪 (图 4A-4B) 对细胞进行24小时的治疗 (或未治疗), CD86 和 MHCII 的表达。此外, 上清液细胞因子阵列 ELISA 法测定了 IL-12p40/70 和 IL-6 的产量 (图 4C-4D)。
中医和 GMPs 在静态状态下表达了很低的 MHC II 类和 CD86 的水平, 这些表达模式在接触 PAMPs 的鸡尾酒时没有显著改变 (图 4A和4B)。同样, 由单核细胞的 MHC II 类的表达也很低, 在玉兰暴露后变化不大 (图 4A)。然而, CD86 在单核细胞中的表达在24小时后, 在24小时的刺激下进一步增加。观察 MoMacs 和 MoDCs 中 MHC II. 类和 CD86 的较高基底表达, 两个种群在玉兰刺激时均表现出强烈的诱导作用。从 MHC II. 类表达后的刺激, 没有统计学差异的中医, GMPs 和单核细胞, 而 MoMacs 和 MoDCs 形成了一个不同的组。然而, 在 CD86 表达方面, 中医和 GMPs 细胞在统计学上不同于 MoMacs 和 MoDCs。然而, 单核细胞与 MoMacs 或 MoDCs 不同。
我们接下来想评估五人口中每一个人的相对能力, 以产生细胞因子的反应 TLR 刺激。因此, 我们在 TLR 激动剂鸡尾酒的存在或缺乏的情况下, 对已排序的人群进行了细胞因子检测。细胞的培养与刺激24小时, 然后上清液收集。我们观察到很少 IL-12p40/70 或 IL-6 生产的中医在 TLR 刺激 (图 4C-4D)。第二个人口 GMPs 无法生产 IL-12p40/70 (图 4C), 但这些细胞在 PAMPs 刺激下产生了少量的 IL-6 (图 4D)。在后三个人群中观察到细胞因子的最高水平。单核细胞和 MoMacs 有非常相似的模式和大小的细胞因子的生产。两者都大大增加了 IL-12p40/70 和适度增加 IL-6 增产刺激 (图 4C-4D)。有趣的是, 在存在 PAMPs MoDCs 没有增加 IL-12p40/70 分泌;然而, 这一人口大大增加了 IL-6 产量 (图 4C-4D)。这些发现表明, 被排序的人群在隔离后维持其免疫功能。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57365/57365fig1.jpg"> 图 1: 前方与侧散射门之间可行的细胞.在转基因脑脊液中培养小鼠骨髓, 利用 7-7-氨基-放线菌 D) 染色后采收后 (PH 值) 和1、3和5天的采收后测定其生存能力。(A) 通过应用门 (可行的) 来选择可行的细胞, 省略了基于 FSC 和 SSC 的小型和高粒度事件。(B) 在可行的 (FSC/SSC) 门内发生的事件产生的 7-反色直方图。阳性的 7-反的事件表明细胞正在凋亡。箭头表示可行的细胞门控。<a href="/files/ftp_upload/57365/57365fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57365/57365fig2.jpg"> 图 2: Ly6C-CD115 人群中的淋巴细胞.采用流式细胞仪对小鼠骨髓进行了 CD3 和淋巴细胞标记 (CD145R、NK1.1) 的培养。(A) 细胞被 Ly6C-PE 和 CD115-APC 染色, 以识别 Ly6C-CD115 细胞。基于单色控制的象限门应用。假彩色0天 (B) CD3、CD45R 和 NK1.1 Ly6C-CD115 细胞的表达, 在收获日 (0 天) 上分析, 直到6天。用流式细胞仪分析软件将细胞计数规范化为模式。箭头指示 Ly6C-CD115 门。<a href="/files/ftp_upload/57365/57365fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57365/57365fig3.jpg"> 图 3: 不同日分选后 Ly6C-CD115+ 细胞的发育动力学.(A) 小鼠骨髓在转基因脑脊液中收获和培养。整除数 1 x 107细胞被收获 (B) 3, (C) 5, 或 (D) 收获后7天 (PH 值)。在指示的天 PH 值, Ly6C-CD115+ 细胞排序从混合的培养 (预先排序) 并且立刻分析后排序 (PS)。用流式细胞仪对细胞进行重组, 对 Ly6C/CD115 表达的变化进行了分析。框和箭头表示排序门。<a href="/files/ftp_upload/57365/57365fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57365/57365fig4.jpg"> 图 4: TLR 刺激后的成熟和细胞因子表达.在 GM-CSF 的3天, 细胞被分类为5个种群。然后他们用 PAMPs 的鸡尾酒治疗 (LPS, 聚 I: C, 和 CpG DNA) 为 24 h. (a) MHC II. 类和 (B) CD86 的平均荧光强度 (), 用流式细胞仪对 PAMPs 进行了后排序 (PS) 和 24 h 的分析。误差条表示3-5 复制实验的标准偏差。(C) IL-12p40/70 和 (D) 从3个共用样品中 IL-6, 用细胞因子阵列斑点印迹酶联免疫吸附法测定了24小时后的上清液和无 PAMPs。RLU相对光单位;++ 表示指定人口的细胞表面标记的存在。<a href="/files/ftp_upload/57365/57365fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td></td>
			<td colspan="2" style="text-align: center;">3天</td>
			<td colspan="2" style="text-align: center;">5天</td>
			<td colspan="2" style="text-align: center;">7天</td>
		</tr>
<tr>
<td>表</td>
			<td style="text-align: left;">单元格类型</td>
			<td style="text-align: center;">分钟</td>
			<td style="text-align: center;">麦克斯</td>
			<td style="text-align: center;">分钟</td>
			<td style="text-align: center;">麦克斯</td>
			<td style="text-align: center;">分钟</td>
			<td style="text-align: center;">麦克斯</td>
		</tr>
<tr>
<td style="text-align: left;">Ly6C-CD115-CD11c-</td>
			<td style="text-align: left;">Cmp</td>
			<td style="text-align: center;">3 x 106
</td>
			<td style="text-align: center;">4 x 106
</td>
			<td style="text-align: center;">5 x 105
</td>
			<td style="text-align: center;">1 x 106
</td>
			<td style="text-align: center;">N/a</td>
			<td style="text-align: center;">N/a</td>
		</tr>
<tr>
<td style="text-align: left;">Ly6C+CD115-</td>
			<td style="text-align: left;">Gmp</td>
			<td style="text-align: center;">2 x 106
</td>
			<td style="text-align: center;">3 x 106
</td>
			<td style="text-align: center;">2 x 106
</td>
			<td style="text-align: center;">3 x 106
</td>
			<td style="text-align: center;">N/a</td>
			<td style="text-align: center;">N/a</td>
		</tr>
<tr>
<td style="text-align: left;">Ly6C+CD115+</td>
			<td style="text-align: left;">单</td>
			<td style="text-align: center;">2 x 106
</td>
			<td style="text-align: center;">3 x 106
</td>
			<td style="text-align: center;">2 x 106
</td>
			<td style="text-align: center;">3 x 106
</td>
			<td style="text-align: center;">5 x 105
</td>
			<td style="text-align: center;">1 x 106
</td>
		</tr>
<tr>
<td style="text-align: left;">Ly6C-CD115+</td>
			<td style="text-align: left;">MoMac</td>
			<td style="text-align: center;">5 x 105
</td>
			<td style="text-align: center;">1 x 106
</td>
			<td style="text-align: center;">3 x 106
</td>
			<td style="text-align: center;">4 x 106
</td>
			<td style="text-align: center;">3 x 106
</td>
			<td style="text-align: center;">4 x 106
</td>
		</tr>
<tr>
<td style="text-align: left;">Ly6C-CD115-CD11c+</td>
			<td style="text-align: left;">MoDC</td>
			<td style="text-align: center;">N/a</td>
			<td style="text-align: center;">N/a</td>
			<td style="text-align: center;">5 x 105
</td>
			<td style="text-align: center;">1 x 106
</td>
			<td style="text-align: center;">3 x 106
</td>
			<td style="text-align: center;">4 x 106
</td>
		</tr>
</tbody></table>表 1: 每 1 x 107个单元格中恢复的最小和最大单元数.CMP (普通髓样祖);GMP (粒细胞/巨噬细胞祖) 单核细胞;MoMac (单核细胞衍生巨噬细胞);MoDC (单核细胞衍生树突状细胞);N/a (不可用);Min (最小细胞产率为 1 x 107细胞);最大 (最大细胞产量从 1 x 107细胞)。

Watch this Scientific Journal Video about Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy at JoVE.com

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

在这里, 我们提供了一种方法, 以识别和隔离大量的 GM-脑脊液驱动髓细胞使用高速细胞排序。根据 Ly6C 和 CD115 的表达, 可以确定五种不同的种群 (常见髓样祖细胞、粒/巨噬细胞祖、单核细胞、单核细胞衍生巨噬细胞和单核细胞衍生的 DCs)。

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

generation-large-numbers-myeloid-progenitors-dendritic-cell

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

9.0K 次观看.  Auburn University. 在这里, 我们提供了一种方法, 以识别和隔离大量的 GM-脑脊液驱动髓细胞使用高速细胞排序。根据 Ly6C 和 CD115 的表达, 可以确定五种不同的种群 (常见髓样祖细胞、粒/巨噬细胞祖、单核细胞、单核细胞衍生巨噬细胞和单核细胞衍生的 DCs)。

视频: Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

科研

生物学

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

小鼠髓质前体 32-脑脊液-R 细胞的增殖和分化

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

发育生物学

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

应用造血干细胞移植评估骨髓增生异常综合征的起源

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

不成熟，成熟和耐受性树突状细胞的生成与代谢不同表型

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

免疫与感染

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

流式细胞术从小鼠组织中鉴定和分离爆破形成单元和集落形成单元红系祖细胞

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

Isolation of Mouse Megakaryocyte Progenitors

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.

小鼠巨核细胞祖细胞的分离

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through ...