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。然而，可重复地从实质中分离造血基质是一项挑战，特别是随着单细胞多组学的出现，这需要高质量的样品制备，同时最大限度地提高基质细胞的产量和多样性。此处描述的方案已经过优化，可与大多数下游多组学应用兼容，同时应对分离造血基质细胞的挑战1,2,19。
分离造血基质细胞时首先要考虑的问题是解离条件。最佳解离对于从组织中释放基质细胞以反映基质区室的完全异质性至关重要 1,2。选择正确的解离混合物取决于实验范围和下游方案。我们建议尽可能新鲜地制备解离混合物，并将它们放在冰上直到使用。有些成分难以溶解，例如分散酶，可能需要更多时间进行移液管混合。不建议涡旋，因为这可能会破坏消化酶的天然构象，最终导致其失活。重要的是，EDTA会抑制酶活性，在主动消化步骤中应避免使用，但如果不能及时去除含有酶缓冲液的EDTA，则可以使用EDTA来停止反应。除了酶混合物本身外，还有两点对于组织的有效解离至关重要，（1）彻底粉碎和切割以增加表面积，以及（2）与消化酶有足够的孵育时间。例如，人类胸腺组织比小鼠组织更难解离，因此消化时间必须更长。如果不对人体组织进行调整，基质产量和细胞多样性将减少（图2A）。解离酶需要 37 °C 的最佳温度，这不可避免地会影响基因表达27。尽管需要冷温或低温解离来保持细胞状态，但目前没有适用于骨骼和骨髓的冷活性解离酶组合。对于任何需要消化的组织，从不同细胞类型获得的异质性和贡献百分比将不可避免地随着消化时间、酶混合物组成和加工而变化。在这些初始步骤的下游，异质性和代表性将进一步受到过滤步骤的限制，例如分选和捕获具有挑战性的细胞类型（例如，来自基质的脂肪细胞）和流体装置通道直径时的喷嘴尺寸。因此，在比较数据集时，我们应该注意，协议中的这些差异会影响亚群的表示。从逻辑上讲，在测量基质系统中扰动的影响时，我们必须保持警惕，保持处理步骤相等。
虽然测序的成本已经并且正在下降，但单细胞技术通常受到可分析细胞数量的限制。为了优化每个试剂盒的容量，尽可能采用造血细胞磁耗竭法。这样可以避免大量的污染细胞，这些细胞在骨髓和胸腺样本组分中都比基质多得多。然而，如果起始材料有限，例如移植后胸腺组织的情况，则磁耗竭策略必然导致的细胞非特异性损失增加，可能使这变得不可取。此外，在组织的制备和消化过程中，细胞外基质成分的粉碎、切割和酶促释放会产生大量碎片。如果将此类污染物留在样品中进行分析，则会导致下游工艺中的堵塞问题，导致单细胞指数分选的负孔，并在分析中严重夸大母门百分比。钙黄绿素染色用于细胞/活细胞的门控阳性。钙黄绿素有不同的颜色，这使得它对您的染色基质具有高度的适应性。使用钙黄绿素时，注意不要使染料反复冻融循环（即，始终新鲜制备并在溶解后保持在室温下）。
基于用 DNA oligos28 条形码编码的抗体对样品进行多路复用的能力提高了 10x Genomics 等平台上单细胞测序反应的实用性。专为样本多路复用而设计的标签抗体对细胞表面分子或核膜具有不同的靶点，对小鼠和人类具有不同的特异性。虽然细胞标签系统已成功用于多种组织，但将消化酶混合物与随后使用标签相结合的实验可能导致回收率大幅降低。我们试图在解离后加入额外的洗涤步骤，并添加EDTA以抑制消化酶的活性。然而，即使采取这些步骤，预计标签识别度也低于造血细胞。结果发现，使用酶消化方案，标签捕获率在30%-70%之间。尽管具有荧光染料偶联物的相同抗体克隆在同一群体中标记了 >90% 的基质细胞，但还是会发生这种情况。任何将组织的酶消化与标签相结合的实验设计都应该进行试点，并谨慎行事以确保酶促抑制。

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，而照射和骨髓移植后4天的胸腺可以低至5,00,000个细胞（图1A）。
细胞毒性调节将优先消融造血细胞，因此基质区室将按比例富集（图1B）。然而，总体细胞密度低将使分离足够数量的胸腺基质细胞变得具有挑战性。因此，可能需要汇集动物以获得与下游多组学应用兼容的细胞数，并尽可能减少材料损失。为了对胸腺基质细胞进行多组学研究，活的、单一的、CD45-Ter119-细胞的荧光激活细胞分选（FACS）（图1C）将确保可以从大多数应激条件下分选足够数量的细胞。
为了保持基质隔室的异质性，我们通常不会对单个阳性基质标记物进行分选，而是排除造血细胞。仍然建议包括阳性基质细胞标志物进行分析，以确保已实现适当的组织解离。然而，FACS分离是不完美的，根据scRNAseq的测定，30%-50%的分选细胞将是造血来源的（图1D，E）。这可以通过在 FACS 之前磁性耗尽 CD45+ 细胞来改善;然而，对于胸腺细胞性降低的条件（图1A），不建议这样做，因为磁耗竭涉及的额外处理会带来材料损失，导致基质产量对于下游应用来说太低。
对于人胸腺基质的分离，最关键的步骤是酶消化。如果匆忙进行解离步骤，则总基质细胞产量以及基质细胞亚型表示将严重降低（图2A）。还建议始终包括胸腺内皮和上皮的标志物，以确保组织消化处于最佳状态，因为简单地对造血标志物进行门控阴性不足以测量实际的基质细胞释放。 如图 2A 所示，其中与孵育 90 分钟的样品相比，消化 30 分钟的样品中造血标志物阴性的细胞百分比更高。然而，如在连续浇口中所见，在较短的消化方案中，内皮细胞和上皮细胞的产量非常差（图2A）。
对于用于多组学应用的人胸腺基质细胞的 FACS 分离，除了泛造血标志物 CD45 和红系标志物 CD235a（图 2B）外，还建议使用造血谱系抗体混合物以及 CD4 和 CD8 抗体进行染色，以改善基质细胞富集。因此，对于胸腺基质的人类多组学分析，应将活的、单一的CD45-CD235a-谱系-CD4-CD8-细胞进行流式细胞仪分选（图2B）。如小鼠样本的scRNAseq所示，这仍然导致30%-50%的造血细胞污染，但这仍然提供了人胸腺基质室的可接受分辨率（图2C，D）。
对于骨骼和 BM 基质，使用与胸腺组织相同的策略;通过对造血标志物阴性的细胞进行门控来分离基质细胞，并包括进一步的基质细胞标志物作为消化对照（图3）。骨骼等钙化组织的解离导致碎片的大量积累。添加钙黄绿素作为活性染料将允许排除大部分此类碎片，否则会错误地夸大排序数量并降低门控群体频率（图3）。这在FACS图中得到了体现，该图比较了所有细胞的门控和仅对Calcein+的门控（图3）。如人胸腺基质细胞所见，造血标志物阴性细胞的百分比不是骨骼和BM组织解离效率的可靠读数（图3）。虽然 CD45-Ter119-门在消化步骤失败的样品中可能含有大量细胞，但对典型骨骼和 BM 基质细胞标志物 CD31、CD140a 和 CD51 的进一步分析清楚地表明基质释放不良（图 3 和 图 4）。
对于小鼠骨骼和BM基质细胞的FACS分离，建议使用如图3所示的门控策略，其中我们仅分选CD45-Ter119-Calcein+，但监测分选群体的前向和侧向散射特性以及基质细胞标志物表达，以确保最佳消化（图3A）。在 FACS 之前，骨髓的造血谱系阳性磁性细胞耗竭和在所示门上分选的结合导致细胞的单细胞测序为 >90% 基质。这种scRNA seq运行的一个例子（图5A，B）说明了在对分析细胞进行基于转录组的注释后，这种低造血污染（造血标记基因的例子，图5C）。这与胸腺基质制备形成鲜明对比，在胸腺基质制备中，由于移植后胸腺组织中的起始材料有限，在分选步骤之前未进行磁性细胞耗竭（图1F）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66231/66231fig01.jpg"> 图 1：小鼠胸腺基质的流式细胞术和单细胞 RNA 测序分析。 （A） 8 周龄小鼠（对照组）、8 周龄小鼠在 4 天前致死照射和移植 4 天（移植）和 2 岁小鼠（老化）中胸腺细胞数量的定量。（B） 代表性流式细胞术图，显示致死照射和移植后 4 天基质细胞亚型的富集。（C） 流式细胞术门控策略，用于分选小鼠胸腺基质，用于单细胞多组学分析。（D） 小鼠胸腺的 UMAP，指示基质（蓝色）和造血细胞（红色）。（E） 胸腺样本中显示鼠造血标记基因的 UMAP。（F） 胸腺 scRNAseq 样品中小鼠基质细胞和造血细胞的定量。 <a href="https://www.jove.com/files/ftp_upload/66231/66231fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66231/66231fig02.jpg"> 图 2：人胸腺基质的流式细胞术和单细胞 RNA 测序分析。 （A） FACS 图显示了不同消化时间对人胸腺基质细胞群释放影响的代表性结果。（B） 流式细胞术门控策略，用于分选人胸腺基质以进行单细胞多组学分析。（C） 人胸腺的 UMAP，指示基质（蓝色）和造血细胞（红色）。（D） 胸腺样本中显示人类造血标记基因的UMAPs。（E） 胸腺 scRNAseq 样品中人基质细胞和造血细胞的定量。 <a href="https://www.jove.com/files/ftp_upload/66231/66231fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66231/66231fig03.jpg"> 图 3：小鼠骨髓基质标志物和钙黄绿素有助于估计解离效率。 （A） 在所有细胞和钙黄绿素+细胞上设门的CD45-Ter119-小鼠骨髓基质细胞的代表性流式细胞术分析。在选择钙黄绿素+细胞时，随后的CD31-门控显示CD140a + CD51+细胞的比例更高。（B） 流式细胞术分析，代表缺失 CD140a+CD51+ 细胞的骨消化失败。<a href="https://www.jove.com/files/ftp_upload/66231/66231fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66231/66231fig04.jpg"> 图 4：骨髓基质门控策略。 （A） 流式细胞术门控策略，用于对小鼠骨髓基质进行单细胞多组学分选。（B） 流式细胞术分析，代表缺失 CD140a+CD51+ 细胞的骨髓消化失败。 <a href="https://www.jove.com/files/ftp_upload/66231/66231fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66231/66231fig05.jpg"> 图 5：骨髓单细胞 RNA 测序与基质细胞和造血细胞组成。 （A） 小鼠骨骼和骨髓的 UMAP，指示基质（蓝色）和造血细胞（红色）。（B） 小鼠骨骼和骨髓样本中基质细胞和造血细胞的定量。（C）小鼠骨髓样本中造血标记基因的UMAP。 <a href="https://www.jove.com/files/ftp_upload/66231/66231fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Stromal Cell Isolation From Hematopoietic Organs

957 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 ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

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 ...