我们得到了马萨诸塞州总医院的HSCI-CRM流式细胞术设施和哈佛大学Bauer Core设施的专家技术支持。T.K 和 K. G 得到了瑞典研究委员会的支持，C.M. 得到了德国研究基金会的支持。我们感谢 Sergey Isaev 和 I-Hsiu Lee 在分析单细胞 RNA 测序数据方面的帮助。

所有与人体组织有关的工作都是在马萨诸塞州总医院内部审查委员会 （IRB） 批准后进行的。所有动物程序均按照马萨诸塞州总医院机构动物护理和使用委员会 （IACUC） 指南进行。本研究采用C57Bl/6只小鼠，年龄8-10周龄，雄性和雌性均为小鼠。这些动物是从商业来源获得的（见 材料表）。
1.鼠胸腺组织的制备
<ol><li>准备以下缓冲液和溶液。<ol><li>培养基 199 （M199） 与 2% （v/v） 胎牛血清 （FBS）（参见 材料表）。</li>
		<li>小鼠解离混合物:0.5 WU/mL Liberase TM 和 6.3 U/mL DNase I，M199 + 2% FBS（参见 材料表）。</li>
		<li>M199 含 2% （w/v） 牛血清白蛋白 （BSA）。 注意:含有FBS的培养基可以切换到BSA以提高RNA质量。</li>
	</ol></li>
	<li>进行小鼠胸腺解剖<ol><li>通过 CO2 窒息对小鼠实施安乐死（遵循机构批准的方案）并将其放在背上。通过喷洒 70% 乙醇弄湿胸部的毛发，然后继续打开胸腔。</li>
		<li>首先用手术剪刀在肋骨下方做一个横向切口，小心地解剖胸腺。切开小鼠两侧的肋骨，一直切到锁骨。然后切开横膈膜，使胸壁可以释放。用一把镊子提起肋骨，露出胸腔。<ol><li>胸腺是一个白色的双叶器官，位于腔体的顶部，就在心脏上方。用镊子握住胸腺，轻轻切开将胸腺固定到位的结缔组织。将解剖的胸腺组织放入冰上含有M199 + 2%FBS的6孔板的孔中。 注意:当处理老年或辐射条件的小鼠或具有胸腺缺陷的突变品系时，胸腺可能非常小，以至于需要解剖显微镜才能可靠地切除器官。</li>
		</ol></li>
		<li>使用解剖显微镜，使用钝头镊子和微弹簧剪刀去除任何胸腺外组织。</li>
	</ol></li>
	<li>进行小鼠胸腺组织的酶解离（30分钟，增量为10分钟）<ol><li>将清洁的胸腺放入 15 mL 管的盖子中，并用无菌手术剪刀将组织细碎。 注意:将用于消化的管帽中的组织切碎可以最大限度地减少由于从一个容器转移到另一个容器而造成的组织损失。当处理老年或辐射条件的小鼠或具有胸腺缺陷的突变菌株时，这一点非常重要。在这些情况下，胸腺体积较小，因此需要尽可能减少物质损失。</li>
		<li>向 15 mL 试管中加入 2 mL 小鼠解离混合物，连接含有胸腺片的盖子，并倒置 5 次以确保组织重新悬浮在解离混合物中。</li>
		<li>为防止泄漏，请用石蜡膜包裹管帽。然后将试管水平放置在37°C的水浴中，以250rpm的转速摇动。孵育 10 分钟。 注:消化方案的成功取决于在37°C和搅拌下进行的孵育。所描述的协议已经使用振荡水浴进行了优化。如果这不可用，在加热环境中启用搅拌的其他方法可能有效，但我们建议首先针对这些设置优化协议。</li>
		<li>将 15 mL 试管放入架子中，让组织碎片沉淀。小心地取出上清液，并将其通过70μm细胞过滤器放入冰冷的50ml管中。</li>
		<li>重复步骤 2-4 两次，总共 3 轮消化。在最后一次孵育结束时，组织应或多或少完全解离。</li>
		<li>向收集的细胞悬液中加入 20 mL M199 + 2% FBS。在4°C下以500× g 旋转5分钟。</li>
		<li>取出上清液并重悬于适当体积的 M199 + 2% FBS 中。然后继续进行细胞计数。 注意:M199 + 2% FBS 重悬的体积将取决于胸腺的状态。对于 6-10 周龄的小鼠，我们建议将组织重悬于 3-4 mL M199 + 2% FBS 中。然而，对于使用老化或辐射条件的组织或具有胸腺缺陷的突变菌株的实验，小胸腺尺寸将需要 0.1-1 mL 的较低体积。</li>
		<li>通过在 0.4% （w/v） 台盼蓝溶液中按 1:2 稀释细胞悬浮液来计数细胞。将细胞加载到血细胞计数器上，并在明场显微镜下计数。对未染色的细胞进行计数，以确定每毫升活细胞的数量，以及蓝色染色的细胞数量，以评估样品的活力。</li>
	</ol></li>
	<li>对小鼠胸腺基质进行荧光激活细胞分选<ol><li>在4°C下以500× g 旋转细胞5分钟。 将细胞以 5 x 107 个细胞/mL 的浓度重悬于补充有 25 U/mL Protector RNase 抑制剂的 M199 + 2% FBS 中（参见 材料表）。</li>
		<li>加入浓度为2μg/ mL的小鼠Fc块（参见 材料表），并在4°C下孵育10分钟。</li>
		<li>用以下抗体染色细胞:CD45-PE / Cy7（4μg/ mL），Ter119-PE（4μg/ mL），CD31-BUV737（2μg/ mL）和EpCam-BV711（2μg/ mL）（参见 材料表）在4°C下30分钟。</li>
		<li>在M199 + 2%BSA中洗涤样品，并在4°C下以500× g 旋转5分钟。</li>
		<li>去除上清液并将细胞重悬于含有活性染料（如DAPI（0.5μg/ mL）或7-AAD（1μg/ mL）的M199 + 2%BSA中（参见 材料表）。按照既定的方案进行胸腺基质细胞的FACS分离19。</li>
	</ol></li>
</ol>2.人胸腺组织的制备
<ol><li>准备以下缓冲液和溶液。<ol><li>培养基 199 （M199），含 2% （v/v） FBS。</li>
		<li>M199 含 1.5% （w/v） BSA。</li>
		<li>人解离混合物:1 mg/ml 胶原酶 IV 和 2 mg/mL 分散酶和 6.3 U/mL DNase I，M199 +1.5% BSA 溶液（参见 材料表）。 注:制备人胸腺组织的最后一个消化步骤包括胰蛋白酶。因此，在此之前的所有步骤都必须在含有 BSA 的缓冲液中进行，因为 FBS 会抑制胰蛋白酶活性。</li>
	</ol></li>
	<li>进行人胸腺组织分离<ol><li>将人胸腺（按照先前发表的方法19收集）置于合适的M199 + 1.5%BSA体积中，并将其储存在冰上直至处理开始。</li>
		<li>在处理人类胸腺组织时，通过在组织培养罩中进行工作并根据 CDC 指南20 使用标准预防措施，将其处理为具有潜在传染性的工作。</li>
		<li>将组织放入装有 10 mL 冰冷 M199 + 1.5% BSA 的 10 cm 培养皿中。使用无菌镊子和剪刀，小心地清除在摘除器官时受损的胸腺外脂肪或组织。</li>
		<li>使用手术剪刀和一把镊子，将胸腺切成 1 cm3 立方体，然后使用 10 mL 注射器的柱塞轻轻向下推到碎片上。这将释放一些发育中的胸腺细胞，从而减少要消化的组织体积。 注意:如果需要对胸腺细胞发育阶段进行后续分析，请保存释放的造血部分。但是，不要将该部分与酶消化过程中产生的基质细胞部分混合。据观察，将两种馏分合并显着增加了细胞悬液中形成堵塞的风险，最终降低了基质细胞的产量。在轻轻粉碎组织的过程中，很少有基质细胞丢失。</li>
	</ol></li>
	<li>进行人胸腺组织的酶解离（90分钟，增量为30分钟）<ol><li>将约 5 mg 轻度压碎的胸腺组织块转移到 50 mL 锥形管的盖子中。然后使用无菌手术剪刀将组织切细。</li>
		<li>将 8 mL 人解离混合物移入 50 mL 试管中，连接含有切碎的人胸腺组织的盖子，然后轻轻倒置试管 5 次，以确保所有组织都与酶混合物混合。</li>
		<li>用石蜡膜固定管帽，防止泄漏。将试管在37°C的振荡（250rpm）水浴中水平孵育30分钟。</li>
		<li>将 50 mL 试管置于架子中，让人胸腺组织碎片沉淀，然后取出上清液并将其通过 70 μm 细胞过滤器放入放置在冰上的新 50 ml 试管中。</li>
		<li>重复步骤2.3.2-1.3.4，用人解离混合物总共进行两轮消化。</li>
		<li>第二轮消化完成后，向剩余组织中再加入 8 mL 人解离混合物。然后将 2 mL 0.25% 胰蛋白酶移液到组织解离管中。在37°C下再孵育30分钟，以250rpm振荡。 注意:人体组织的消化时间至少为 90 分钟是绝对必要的，因为较短的孵育时间会导致基质细胞产量急剧降低。</li>
		<li>完成最后一个孵育步骤后，通过70μm细胞过滤器，将细胞悬浮液和剩余的组织片段与先前步骤中收集的上清液混合在一起。向样品中加入 3 mL FBS 以破坏胰蛋白酶反应并置于冰上。</li>
		<li>继续按照步骤 1.3 中所述对细胞进行计数。</li>
	</ol></li>
	<li>对人胸腺基质进行荧光激活细胞分选<ol><li>旋转人胸腺的细胞悬浮液，并以 5 x 107 个细胞/mL 的浓度重悬于补充有 25 U/mL Protector RNase 抑制剂的 M199 + 2% FBS 中。</li>
		<li>与人Fc模块（5μg/ mL）（参见 材料表）在4°C下孵育10分钟。</li>
		<li>用CD45-BV711（2.5μg/ mL），CD235a-BV711（2.5μg/ mL），Lineage-cocktail-FITC（3.76μg/ mL），CD66b-FITC（20μL / 100μL样品），CD8-APC / Cy7（5μL / 100μL样品），CD4-BV605（5μL / 100μL样品），CD31-PE / Dazzle594（10μg/ mL）和EpCam-BV421（2.5μg/ mL）（参见 材料目录）染色30分钟。</li>
		<li>在M199 + 1.5%BSA中洗涤样品，并在4°C下以500× g 旋转5分钟。</li>
		<li>去除上清液并将细胞重悬于含有活性染料（如DAPI（0.5μg/ mL）或7-AAD（1μg/ mL）的M199 + 1.5%BSA中。按照既定的方案进行胸腺基质细胞的FACS分离19。</li>
	</ol></li>
</ol>3.鼠骨和骨髓组织的制备
注:骨和骨髓部分在两个单独的消化反应中制备，以获得基质细胞的最大纯度和组织的最佳解离。在酶解步骤之后，可以将样品合并为一个基质室进行分类。
<ol><li>准备以下缓冲液和溶液。<ol><li>M199 与 2% （v/v） FBS。</li>
		<li>小鼠 B&M（骨和骨髓）解离混合物:M199 +2% FBS 中的 Stemxyme 2 mg/mL、分散酶 1 mg/mL 和 10 U/mL DNase I（参见 材料表）。 注意:准备新鲜的 B&M 解离混合物，并将混合物放在冰上直至使用。</li>
		<li>M199 含 0.5% （w/v） BSA。</li>
		<li>含 0.5% （w/v） BSA 的磷酸盐缓冲盐水 （PBS）。</li>
		<li>M199 含 2% （w/v） 牛血清白蛋白 （BSA） 和 2 mM EDTA。 注:含有FBS的培养基可以切换到BSA以提高RNA质量。</li>
	</ol></li>
	<li>进行小鼠骨髓分离<ol><li>通过 CO2 窒息对小鼠实施安乐死（遵循机构批准的方案）。喷洒 70% 乙醇以弄湿毛皮。</li>
		<li>小心地从两侧取出股骨，胫骨，骨盆和肱骨，并将它们放入含有M199 + 2%FBS的冰21,22孔中。</li>
		<li>用组织湿巾去除所有肌肉、韧带和软骨组织，以尽量减少对骨骼的损伤并保留骨膜层。将它们放入冰上含有 M199 + 2% FBS 的新孔中。</li>
		<li>用手术刀切开长骨的生长板，切去骨骺，将其放入10厘米的培养皿中，在冰上加入M199 + 2%FBS。</li>
		<li>将骨髓从骨头中冲出，直到呈淡白色。使用 28 G 针头和装满 10 mL M199 + 2% FBS 的 10 mL 注射器将骨髓冲洗到冰上的 15 mL 管中。将冲洗的骨头与骨骺一起放入 10 厘米的培养皿中。 注意:完全冲洗骨髓以完全分离组织将导致更好的样品制备。</li>
	</ol></li>
	<li>进行小鼠骨髓组织的酶解离（总共 30 分钟，增量为 10 分钟）<ol><li>将 15 mL 试管直立在冰上，直到骨髓沉入试管底部。通过移液缓慢去除 10 mL M199 + 2% FBS。</li>
		<li>向15mL管中加入4mL B&M解离混合物，并将其置于37°C水浴（不摇晃）中10分钟。</li>
		<li>5分钟后，将试管倒置三次，然后将其放回水浴中。</li>
		<li>第一个 10 分钟后，确保沉淀位于底部并收集上清液，同时通过 70 μm 细胞过滤器过滤到冰冷的 50 mL 管中。将 2 mL 新鲜的 B&M 解离混合物加入剩余的 BM 沉淀中，然后将其放回水浴中。</li>
		<li>通过使用 1 mL 移液管剧烈移液来解离任何剩余的骨髓碎片。将所有产生的细胞悬液收集在同一管中，就像在前面的步骤一样。向收集的细胞悬液中加入 20 mL M199 + 0.5% BSA + 2 mM EDTA。 注:在步骤3.3.5中，在最终消化样品后添加的缓冲液含有EDTA，以帮助抑制进一步的酶活性。</li>
	</ol></li>
	<li>对小鼠骨髓组织进行造血细胞耗竭。<ol><li>在4°C下以500× g 离心细胞5分钟。 用靶向 CD45、Ter119、CD11b、Gr1、B220 和 CD3 的生物素抗体 （5 mg/mL） 重悬于 500 μL PBS + 0.5% BSA 中（参见 材料表）。转移到带盖的圆底管中。</li>
		<li>在室温下，在黑暗中在轨道振荡器上孵育10分钟。</li>
		<li>彻底涡旋磁珠，并向试管中加入 25 μL 磁珠。</li>
		<li>在室温下，在黑暗中在轨道振荡器上孵育5分钟。</li>
		<li>将试管放入匹配的磁铁中 2-3 分钟，并将上清液收集在新鲜的试管中。 注意:在此步骤之后，骨髓可以合并或与骨组分开。如果谱系去除成功，则所得细胞悬液应不含红细胞。</li>
	</ol></li>
	<li>进行小鼠骨组织的酶解离。<ol><li>用 50 mL 管的扁平盖端将骨头在 10 cm 培养皿中切成小块。通过70μm细胞过滤器过滤上清液，以分离过滤器内的骨碎片。 注意:或者，可以使用研钵和研杵进行骨碎裂。然而，当骨头被磨碎而不是破碎时，这种选择往往会产生更多的碎片和更低的基质细胞活力。基质细胞仍然附着在骨骼上和骨骼中，含有造血细胞的上清液应被丢弃。</li>
		<li>用剪刀剪开细胞过滤器中的骨头碎片。这将增加表面积，从而实现更有效的酶解离。用 5 mL M199 + 2% FBS 清洗骨头。</li>
		<li>将骨部分放入 5 mL 小鼠 B&M 解离液（参见 材料表）混合物中，放入新的 50 mL 管中。</li>
		<li>为防止泄漏，请用石蜡膜包裹管帽。然后将试管水平置于37°C水浴中，以120rpm振荡30分钟。</li>
		<li>消化后，加入 25 mL M199 + 0.5% BSA + 2 mM EDTA，并通过 70 μm 细胞过滤器过滤含有基质细胞的上清液到冰冷的 50 ml 管中。 注意:检查产生的骨头碎片是否因消化而明显减少。在步骤3.5.5中，在最终消化样品后添加的缓冲液含有EDTA，以帮助抑制进一步的酶活性。</li>
	</ol></li>
	<li>对小鼠骨骼和骨髓基质进行荧光激活细胞分选。<ol><li>在4°C下以500× g 旋转细胞5分钟。</li>
		<li>以10μg/ mL的浓度在PBS + 0.5%BSA + 2mM EDTA中重悬于小鼠Fc块中，并在4°C下孵育10分钟。</li>
		<li>用以下抗体对细胞（多达 1000 万个细胞/100 μL 总染色体积）进行染色:CD45-PE/Cy7 （1 μg/mL）、Ter119-PE/Cy7 （1 μg/mL）、CD31-BV421 （0.66 μg/mL）、CD140a-APC （2 μg/mL）、Sca1-AF700 （0.66 μg/mL）、CD51-PE （2 μg/mL）、CD105-PE/dazzle （2 μg/mL）（参见 材料表）。</li>
		<li>让Cal黄绿素-AM平衡至室温，并重悬于50μL DMSO中。为每次使用准备新鲜的。重悬后在室温下保存。在加入已经重悬于抗体染色混合物中的样品（20μg/ mL）之前，在PBS（0.1mg / mL）中预稀释钙黄绿素，并在4°C下在黑暗中孵育20-30分钟。</li>
		<li>在PBS + 0.5%BSA + 2mM EDTA中洗涤样品，并在4°C下以500× g 离心5分钟。</li>
		<li>取出上清液并将细胞重悬于PBS + 0.5%BSA + 2mM EDTA中。按照既定的方案进行骨和骨髓基质细胞的FACS分离19。</li>
	</ol></li>
</ol>

优化从胸腺和骨髓中分离基质细胞

<ol>
	<li>Baryawno, N., 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+cellular+taxonomy+of+the+bone+marrow+stroma+in+homeostasis+and+leukemia.">A cellular taxonomy of the bone marrow stroma in homeostasis and leukemia.</a> Cell. 177 (7), 1915-1932 (2019).</li><li>Severe, N., 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=Stress-induced+changes+in+bone+marrow+stromal+cell+populations+revealed+through+single-cell+protein+expression+mapping.">Stress-induced changes in bone marrow stromal cell populations revealed through single-cell protein expression mapping.</a> Cell Stem Cell. 25 (4), 570-583 (2019).</li><li>Han, J., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+2020+view+of+thymus+stromal+cells+in+t+cell+development.">A 2020 view of thymus stromal cells in t cell development.</a> J Immunol. 206 (2), 249-256 (2021).</li><li>Park, J. E., 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+cell+atlas+of+human+thymic+development+defines+t+cell+repertoire+formation.">A cell atlas of human thymic development defines t cell repertoire formation.</a> Science. 367 (6480), (2020).</li><li>Denisenko, E., 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=Systematic+assessment+of+tissue+dissociation+and+storage+biases+in+single-cell+and+single-nucleus+rna-seq+workflows.">Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus rna-seq workflows.</a> Genome Biol. 21 (1), 130 (2020).</li><li>Lischetti, U., 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=Dynamic+thresholding+and+tissue+dissociation+optimization+for+cite-seq+identifies+differential+surface+protein+abundance+in+metastatic+melanoma.">Dynamic thresholding and tissue dissociation optimization for cite-seq identifies differential surface protein abundance in metastatic melanoma.</a> Commun Biol. 6 (1), 830 (2023).</li><li>Ding, L., Saunders, T. L., Enikolopov, G., Morrison, 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=Endothelial+and+perivascular+cells+maintain+haematopoietic+stem+cells.">Endothelial and perivascular cells maintain haematopoietic stem cells.</a> Nature. 481 (7382), 457-462 (2012).</li><li>Mendez-Ferrer, 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=Mesenchymal+and+haematopoietic+stem+cells+form+a+unique+bone+marrow+niche.">Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.</a> Nature. 466 (7308), 829-834 (2010).</li><li>Raaijmakers, M. H., 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=progenitor+dysfunction+induces+myelodysplasia+and+secondary+leukaemia.">progenitor dysfunction induces myelodysplasia and secondary leukaemia.</a> Nature. 464 (7290), 852-857 (2010).</li><li>Himburg, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+bone+marrow+sources+of+pleiotrophin+control+hematopoietic+stem+cell+maintenance+and+regeneration.">Distinct bone marrow sources of pleiotrophin control hematopoietic stem cell maintenance and regeneration.</a> Cell Stem Cell. 23 (3), 370-381 (2018).</li><li>Zhou, B. O., 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=marrow+adipocytes+promote+the+regeneration+of+stem+cells+and+haematopoiesis+by+secreting+scf.">marrow adipocytes promote the regeneration of stem cells and haematopoiesis by secreting scf.</a> Nat Cell Biol. 19 (8), 891-903 (2017).</li><li>Steinmann, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+human+thymus+during+aging.">Changes in the human thymus during aging.</a> Curr Top Pathol. 75, 43-88 (1986).</li><li>Steinmann, G. G., Klaus, B., Muller-Hermelink, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+involution+of+the+ageing+human+thymic+epithelium+is+independent+of+puberty.+A+morphometric+study.">The involution of the ageing human thymic epithelium is independent of puberty. A morphometric study.</a> Scand J Immunol. 22 (5), 563-575 (1985).</li><li>Ambrosi, T. H., 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=Adipocyte+accumulation+in+the+bone+marrow+during+obesity+and+aging+impairs+stem+cell-based+hematopoietic+and+bone+regeneration.">Adipocyte accumulation in the bone marrow during obesity and aging impairs stem cell-based hematopoietic and bone regeneration.</a> Cell Stem Cell. 20 (6), 771-784 (2017).</li><li>Ho, Y. H., 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=Remodeling+of+bone+marrow+hematopoietic+stem+cell+niches+promotes+myeloid+cell+expansion+during+premature+or+physiological+aging.">Remodeling of bone marrow hematopoietic stem cell niches promotes myeloid cell expansion during premature or physiological aging.</a> Cell Stem Cell. 25 (3), 407-418 (2019).</li><li>Kusumbe, A. 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=Age-dependent+modulation+of+vascular+niches+for+haematopoietic+stem+cells.">Age-dependent modulation of vascular niches for haematopoietic stem cells.</a> Nature. 532 (7599), 380-384 (2016).</li><li>Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+enzymes+improve+isolation+and+characterization+of+the+adult+thymic+epithelium.">Purified enzymes improve isolation and characterization of the adult thymic epithelium.</a> J Immunol Methods. 385 (1-2), 23-34 (2012).</li><li>Stoeckle, 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=Isolation+of+myeloid+dendritic+cells+and+epithelial+cells+from+human+thymus.">Isolation of myeloid dendritic cells and epithelial cells from human thymus.</a> J Vis Exp. (79), e50951 (2013).</li><li>Gustafsson, K., Scadden, D. T. <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+thymus+stromal+cells+from+human+and+murine+tissue.">Isolation of thymus stromal cells from human and murine tissue.</a> Methods Mol Biol. 2567, 191-201 (2023).</li><li>Amend, S. R., Valkenburg, K. C., Pienta, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+hind+limb+long+bone+dissection+and+bone+marrow+isolation.">Murine hind limb long bone dissection and bone marrow isolation.</a> J Vis Exp. (110), e53936 (2016).</li><li>Zhu, H., 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+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+compact+bone.">A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone.</a> Nat Protoc. 5 (3), 550-560 (2010).</li><li>Calvi, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche.">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425 (6960), 841-846 (2003).</li><li>Kode, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukaemogenesis+induced+by+an+activating+beta-catenin+mutation+in+osteoblasts.">Leukaemogenesis induced by an activating beta-catenin mutation in osteoblasts.</a> Nature. 506 (7487), 240-244 (2014).</li><li>Agarwal, 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=Mesenchymal+niche-specific+expression+of+cxcl12+controls+quiescence+of+treatment-resistant+leukemia+stem+cells.">Mesenchymal niche-specific expression of cxcl12 controls quiescence of treatment-resistant leukemia stem cells.</a> Cell Stem Cell. 24 (5), 769-784 (2019).</li><li>Duarte, 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=Inhibition+of+endosteal+vascular+niche+remodeling+rescues+hematopoietic+stem+cell+loss+in+aml.">Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in aml.</a> Cell Stem Cell. 22 (1), 64-77 (2018).</li><li>O'flanagan, C. H., 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=Dissociation+of+solid+tumor+tissues+with+cold+active+protease+for+single-cell+rna-seq+minimizes+conserved+collagenase-associated+stress+responses.">Dissociation of solid tumor tissues with cold active protease for single-cell rna-seq minimizes conserved collagenase-associated stress responses.</a> Genome Biol. 20 (1), 210 (2019).</li><li>Stoeckius, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+hashing+with+barcoded+antibodies+enables+multiplexing+and+doublet+detection+for+single+cell+genomics.">Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics.</a> Genome Biol. 19 (1), 224 (2018).</li></ol>

D.T.S. 是 Agios Therapeutics 和 Editas Medicines 的董事和股东;Magenta Therapeutics 和 LifeVault Bio 的创始人、董事、股东和科学顾问委员会成员，Fate Therapeutics 和 Garuda Therapeutics 的股东和创始人，Clear Creek Bio 的董事、创始人和股东，FOG Pharma、Inzen Therapeutics 和 VCanBio 的顾问，以及 Sumitomo Dianippon 赞助研究资金的接受者。D.T.S和K.G是专利US20220143099A1的发明者。

造血器官中的基质细胞对于正常造血至关重要，而造血基质扰动可导致造血维持和对应激反应的严重损害 9,23,24。深入了解造血基质细胞对于理解血液疾病至关重要 7,9,10,24 以及开发对抗它们的治疗方法的能力 25,26</sup...

在健康的成年人中，血细胞的从头产生发生在骨髓和胸腺中。这些部位的基质细胞对于维持造血至关重要，但基质占组织的不到 1%，2,3,4。因此，获得支持基质的造血干细胞的纯分离株是一个重大挑战，特别是对于单细胞多组学而言，需要权宜之计才能获得高质量的样本。不同消化混合物的成分可能会干扰多组学分析中的某些步骤5,6。本文介绍的方案详细介绍了从骨髓和胸腺组织中分离出多种基质细胞的方法。
骨髓和胸腺中基质成分的扰动会导致血细胞发育的严重破坏，并可能导致恶性肿瘤 7,8,9。在细胞毒性调理和骨髓移植后，支持基质的造血功能受到损害，导致维持造血干细胞和祖细胞 （HSPC） 的细胞因子和生长因子的分泌减少2,10,11。此外，衰老会影响骨髓和胸腺基质细胞，可能导致造血表型老化。胸腺是第一个经历广泛的年龄相关退化的器官。早在青春期开始时，脂肪和纤维化组织就开始取代 T 细胞支持基质12,13。在骨髓中，脂肪细胞含量随着年龄的增长而增加，血管和骨内膜壁龛显着重塑14,15,16。
为了能够在多种应激状态下以及人和小鼠组织的胸腺中研究造血支持基质，我们优化了先前发表的消化方案1、2、8、17、18。这些方案确保了细胞的高效和可重复的分离，并且它们与单细胞RNA测序（scRNAseq）和其他类型的多组学兼容。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>7AAD (7-aminoactinomycin D)</td>
			<td>BD Biosciences</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human Lineage Cocktail 3-FITC</td>
			<td>BD Biosciences</td>
			<td>643510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Millipore Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>C57Bl/6 mice</td>
			<td>Jackson</td>
			<td>664</td>
			<td>Males or females, 8-12 weeks old</td>
		</tr>
		<tr>
			<td>Calcein&#160;</td>
			<td>Fisher Scientific</td>
			<td>65-0853-78</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Millipore Sigma</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Sterile Cell Strainers, White, Mesh Size: 70 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dilactate)</td>
			<td>Biolegend</td>
			<td>422801</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Thermo Fisher Scientific</td>
			<td>17105041</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>90083</td>
			<td>&#160;2500 U/mL</td>
		</tr>
		<tr>
			<td>Easysep mouse streptavidin RapidSpheres Isolation kit</td>
			<td>StemCell Technologies</td>
			<td>19860</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>A31605-01</td>
			<td>Qualified One Shot</td>
		</tr>
		<tr>
			<td>Human Fc Block</td>
			<td>BD Biosciences</td>
			<td>564220</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TM&#160;</td>
			<td>Millipore Sigma</td>
			<td>5401127001</td>
			<td>Research Grade</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Gibco</td>
			<td>12350</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD235a-BV77</td>
			<td>BD Biosciences</td>
			<td>740785</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD31-PE/Dazzle594</td>
			<td>Biolegend</td>
			<td>303130</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD45-BV77</td>
			<td>Biolegend</td>
			<td>304050</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD4-BV605</td>
			<td>BD Biosciences</td>
			<td>562658</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD66b-FITC</td>
			<td>BD Biosciences</td>
			<td>555724</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human CD8-APC/Cy7</td>
			<td>BD Biosciences</td>
			<td>557760</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-human EpCam-BV421</td>
			<td>Biolegend</td>
			<td>324220</td>
			<td></td>
		</tr>
		<tr>
			<td>Protector RNase Inhibitor</td>
			<td>Millipore Sigma</td>
			<td>3335402001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD105-PE /dazzle594</td>
			<td>Biolegend</td>
			<td>120424</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD11b-Biotin</td>
			<td>Biolegend</td>
			<td>101204</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD140a-APC</td>
			<td>Fisher Scientific</td>
			<td>17-1401-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD31-BUV737</td>
			<td>BD Biosciences</td>
			<td>612802</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD31-BV421</td>
			<td>Biolegend</td>
			<td>102424</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD3-Biotin</td>
			<td>Biolegend</td>
			<td>100244</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45.2-Biotin</td>
			<td>Biolegend</td>
			<td>109804</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45-PE/Cy7</td>
			<td>Biolegend</td>
			<td>103114</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45-PE/Cy7</td>
			<td>Biolegend</td>
			<td>103114</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45R/B220-Biotin</td>
			<td>Biolegend</td>
			<td>103204</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD51-PE</td>
			<td>Biolegend</td>
			<td>104106</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse EpCam-BV711</td>
			<td>BD Biosciences</td>
			<td>563134</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ly-6A/E(Sca-1)-AF700</td>
			<td>Biolegend</td>
			<td>108142</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ly-6G/Ly-6C(Gr1)-Biotin</td>
			<td>Biolegend</td>
			<td>108404</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ter119-Biotin</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ter119-PE</td>
			<td>Biolegend</td>
			<td>116208</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ter119-PE/Cy7</td>
			<td>Biolegend</td>
			<td>116222</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemxyme&#160;</td>
			<td>Worthington Biochemical</td>
			<td>LS004107</td>
			<td></td>
		</tr>
	</tbody>
</table>

stromal cell isolation from hematopoietic organs

单细胞测序使得造血器官基质中异质细胞群的定位成为可能。这些方法提供了一个视角，通过它来研究以前未解决的稳态异质性，以及由外在应激或衰老过程中诱导的细胞类型表示的变化。在这里，我们提出了从小鼠和人胸腺以及小鼠骨骼和骨髓中分离高质量基质细胞群的逐步方案。通过这些方案分离的细胞适用于生成高质量的单细胞多组学数据集。本文将讨论样本消化、造血谱系耗竭、FACS分析/分选的影响，以及这些因素如何影响与单细胞测序的兼容性。通过FACS谱的示例，表明测序后分析中成功和低效的解离以及下游基质细胞产量，为用户提供了可识别的指针。考虑基质细胞的特定要求对于获得高质量和可重复的结果至关重要，这些结果可以推动该领域的知识发展。

这些方案从胸腺和骨髓中产生可重复的基质细胞品种，适用于流式细胞术分析，以及单细胞多组学，如scRNA测序。小鼠胸腺组织在应激源的作用下经历了显著的重塑，例如骨髓移植前的细胞毒性调节或自然衰老过程。因此，在这两种情况下，胸腺细胞性都急剧降低（图1A）。虽然来自8周大的野生型小鼠的胸腺含有大约1亿个细胞，但2岁小鼠的细胞密度可以预期是其中的一半，...

Watch this Scientific Journal Video about Stromal Cell Isolation From Hematopoietic Organs at JoVE.com

作者聚焦:使用基质细胞分离进行单细胞测序推进造血研究

在这里，我们提出了能够从小鼠骨骼、骨髓、胸腺和人胸腺组织中分离基质细胞的方案，这些组织与单细胞多组学兼容。

从造血器官中分离基质细胞

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

骨髓和胸腺中血细胞的发育高度依赖基质细胞的支持。造血基质细胞功能的扰动可导致血液生成效率低下甚至恶性。我们的实验室专注于了解造血的基质细胞调节以及如何操纵它以获得治疗效果。单细胞测序的出现推动了许多研究领域的最新进展。在我们的领域也是如此，单细胞分辨率使得能够识别出以前未知的造血器官中的异质基质细胞群。我们的协议已经过优化以解决这个问题，我们希望它们能够对基质细胞进行更多的研究，以解决该领域的关键问题。

stromal-cell-isolation-from-hematopoietic-organs

Research

JoVE Journal

Immunology and Infection

Stromal Cell Isolation From Hematopoietic Organs

928 次观看.  Massachusetts General Hospital. 骨髓和胸腺中血细胞的发育高度依赖基质细胞的支持。造血基质细胞功能的扰动可导致血液生成效率低下甚至恶性。我们的实验室专注于了解造血的基质细胞调节以及如何操纵它以获得治疗效果。单细胞测序的出现推动了许多研究领域的最新进展。在我们的领域也是如此，单细胞分辨率使得能够识别出以前未知的造血器官中的异质基质细胞群。我们的协议已经过优化以?...

视频: 作者聚焦:使用基质细胞分离进行单细胞测序推进造血研究

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

集落形成细胞（CFC）的含量对人体的造血细胞

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

科研

免疫与感染

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

从小鼠多个器官分离和定量寨卡病毒

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

从人类和小鼠心肌标本中分离巨噬细胞子集和细胞位细胞

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Isolation and Quantification of Epstein-Barr Virus from the P3HR1 Cell Line

The Epstein-Barr virus (EBV), formally designated as Human herpesvirus 4 (HHV-4), is the first isolated human tumor virus. Nearly 90-95% of the world&#39;s adult population is infected by EBV. With the recent advancements in molecular biology and immunology, the application of both in vitro and in vivo experimental models has provided deep and meaningful insight into the pathogenesis of EBV in many diseases as well as into EBV-associated tumorigenesis. The aim of this visualized experiment paper is to provide an overview of the isolation of EBV viral particles from cells of the P3HR1 cell line, followed by quantification of the viral preparation. P3HR1 cells, originally isolated from a human Burkitt lymphoma, can produce a P3HR1 virus, which is a type 2 EBV strain. The EBV lytic cycle can be induced in these P3HR1 cells by treatment with phorbol 12-myristate 13-acetate (PMA), yielding EBV viral particles.
Using this protocol for the isolation of EBV particles, P3HR1 cells are cultured for 5 days at 37 &#176;C and 5% CO2 in complete RPMI-1640 medium containing 35 ng/mL PMA. Subsequently, the culture medium is centrifuged at a speed of 120 x g for 8 min to pellet the cells. The virus-containing supernatant is then collected and spun down at a speed of 16,000 x g for 90 min to pellet the EBV particles. The viral pellet is then resuspended in a complete RPMI-1640 medium. This is followed by DNA extraction and quantitative real-time PCR to assess the concentration of EBV particles in the preparation.

This protocol permits the isolation of Epstein-Barr virus particles from the human P3HR1 cell line upon inducing the viral lytic cycle with phorbol 12-myristate 13-acetate. DNA is subsequently extracted from the viral preparation and subjected to real-time PCR to quantify the viral particle concentration.