我们要感谢 J. Gaar 和 m. Vroomen (荷兰马斯特里赫特大学) 的技术支持。此外, 我们还要感谢 k. Verboven、汉森、j. Jocken 和 e Blaak 提供用于建立本议定书和随后的实验的血液和组织活检。

根据《赫尔辛基宣言》, Hasselt 和比利时 Hasselt 大学医学伦理委员会 Jessa 医院批准的研究对象, 从样本中抽取内脏和皮下组织。
1. 试剂的制备
<ol><li>胶原酶溶液<ol>
<li>溶解1克胶原酶 I 在10毫升磷酸盐缓冲盐水 (PBS, 没有钙和镁) 做100毫克/毫升存货解答。准备200µL 整除数和存储在-20 摄氏度。</li>
		<li>在10毫升的 PBS 中溶解1克胶原酶, 制作100毫克/毫升的库存溶液。准备200µL 整除数和存储在-20 摄氏度。</li>
		<li>溶解10毫克的 DNase I 在10毫升的 PBS, 使10毫克/毫升的库存解决方案。准备180µL 整除数和存储在-20 摄氏度。</li>
		<li>添加100µL 胶原酶 I (100 毫克/毫升), 100 µL 胶原酶 XI (100 毫克/毫升), 90 µL DNase I (10 毫克/毫升) 10 毫升 DMEM 火腿的 F12。使胶原酶溶液新鲜的每一个隔离。</li>
	</ol>
</li>
	<li>红细胞溶解缓冲液<ol>
<li>在100毫升超纯水中溶解0.84 克 NH4Cl。</li>
		<li>使用前将 pH 值设置为7.4。在4摄氏度的玻璃瓶中贮存。</li>
		<li>使用前将红细胞溶解缓冲剂放在冰上。</li>
	</ol>
</li>
	<li>缓冲区<ol>
<li>在100毫升的 pbs 中溶解0.5 克牛血清白蛋白 (bsa), 以获得 0.5% BSA pbs。</li>
		<li>在100毫升 0.5% BSA pbs 中溶解65毫克的 nan3以获得 10 mM 南3 0.5% BSA pbs。在4摄氏度的玻璃瓶中贮存溶液。</li>
		<li>在使用前, 在冰上放置一个缓冲器。 警告: NaN3具有剧毒。在处理 NaN3时, 在通风罩中工作, 戴上安全眼镜和防护手套。</li>
	</ol>
</li>
	<li>人 IgG 阻滞<ol>
<li>在10毫升 PBS 中溶解10毫克的人体 IgG 以获得1毫克/毫升。准备100µL 整除数和存储在-20 摄氏度。</li>
		<li>使用前将人体 IgG 块放在冰上。</li>
	</ol>
</li>
</ol>2. SVF 的隔离
<ol><li>切割1克的活检成小块 (2 毫米2) 与手术刀和转移到50毫升离心管 (例如, 猎鹰管)。在样品中加入10毫升胶原酶溶液。 注: 完全关闭管盖, 并打开盖子¼回。</li>
	<li>在37摄氏度的水浴中孵育60分钟, 在柔和的震动下 (60 圈/分钟)。</li>
	<li>用200µM 过滤器过滤产生的悬浮液, 并在新的50毫升离心管中收集样品。在过滤器顶部添加7毫升 PBS, 冲洗过滤器并获得所有电池。</li>
	<li>离心样品在 280 x g 5 分钟在4°c。</li>
	<li>通过吹打去除漂浮脂肪细胞的分数。细胞颗粒是 SVF。 注: 取出脂肪细胞分数以获得 SVF。避免淹没在样本中的整个尖端, 因为这将只删除 PBS, 而不是浮动脂肪细胞。</li>
	<li>并用重悬在5毫升的 PBS 中 SVF 去除胶原酶, 用70µM 过滤器过滤悬浮液, 用5毫升 PBS 冲洗过滤器, 并将样品离心在 280 x g 处, 以5摄氏度为4分钟。</li>
	<li>去除3毫升红细胞溶解缓冲液中的上清和并用重悬颗粒。</li>
	<li>在冰上孵化5分钟。孵化后添加7毫升 PBS。</li>
	<li>离心样品在 280 x g 5 分钟在4°c。</li>
</ol>3. 流式细胞仪分析中的 SVF 染色
<ol><li>将细胞颗粒溶于90µL 4 °c 的内流器缓冲液中, 加入10µL 1 毫克/毫升人 IgG 阻滞剂。在 96 v 型井板的2口井中划分细胞悬浮。将盘子放在冰上, 让人体 IgG 块孵化15分钟。</li>
	<li>在每个样品上添加100µL 的流值器缓冲器, 以5分钟为 280 x 克, 在4摄氏度的情况下清洗和离心板材。在不攻板的情况下, 将盘子倒在一个平滑的运动中, 去掉上清。 注意: 一定要用纸巾把盘子顶部的剩余液体移除, 同时保持盘子颠倒。</li>
	<li>如表 1和表 2中所述, 为巨噬细胞和 DC 子集 (1) 和 T 和 B 单元格子集 (资产管制面板 2) 准备抗体鸡尾酒。在优化抗体浓度后选择了表 1和表 2中描述的卷, 并且足够用于一个增值税或 sAT 示例。 注意: 在 "流控制面板 1" 中, 使用标记 CD303 和 CD141 确认 CD11C+ CD11B低单元格是 dc。但是, 建议将这些标记排除在面板中, 以包括活/死染色。当 PE 通道不使用时, 1 和2都可以与活/死可修复的红死细胞染色试剂盒的生存性染色结合在一起, 因为在面板1中排除 CD303。根据制造商的指示执行生存能力染色。</li>
	<li>并用重悬在29.5 µL 抗体鸡尾酒中的球团, 1 和23µL 抗体鸡尾酒为外地资产管制小组2。在黑暗中的冰上孵化30分钟。</li>
	<li>向每个井中添加150µL 的操作器缓冲区, 并并用重悬单元颗粒以执行第二次清洗步骤。离心板为5分钟在 280 x g 和4°c。把盘子倒过来, 去掉上清。</li>
	<li>在每个井中添加150µL 1% 甲醛溶液来修复细胞。通过吹打与 P200 吸管将细胞悬浮液从各井转移到相应的转流管。在4摄氏度的7天的黑暗中存储资产管制管道。 注: 直接测量也是可能的。在每个井中添加150µL 的吹打, 而不是1% 甲醛, 用 P200 吸管将细胞转移到相应的流化管, 并对细胞进行分析。 注意: 甲醛是非常有毒的。在通风罩中工作时, 要准备甲醛溶液, 避免吸入, 戴上手套和安全眼镜保护。</li>
</ol>4. 流式细胞仪分析
<ol><li>在第一次测量之前, 使用无瑕负控制来设置正散射 (FSC) 和侧散射 (SSC)。根据制造商的指示调整流式细胞仪的电压, 使所有感兴趣的人群都能在 FSC 和 SSC 图中可见, 并且可以区分碎片和活细胞。</li>
	<li>根据制造商的协议, 使用抗体捕捉珠进行多色补偿分析。</li>
	<li>制备荧光减一 (FMO) 控制通过使抗体组合, 但排除一个抗体从混合。对每种抗体都做到这一点, 为1和8抗体混合, 为外地资产管制组2提供6抗体混合。这些 FMO 抗体混合用于染色 SVF, 如上文所述的协议。</li>
	<li>测量所有 FMO 控制, 并设置基于 FMO 控制的浇口策略。使用 FMO 控件检测单元格的可能的自动荧光。 注意: 通过从混合中移除一个抗体, 在该通道中检测到的任何荧光电平都是背景/autofluorescent 信号。因此, 通过比较不同的 FMO 控制外地观察结果, 门可以吸引特定的人群, 以确保 gatings 是基于阳性细胞, 而不是基于自动荧光。</li>
	<li>在将其放进流式细胞仪并开始测量之前, 将 800 rpm 的能量计管涡旋。 注意: 建议在活门中至少有5万个事件, 以确保从每个亚群中测量出足够的细胞。</li>
</ol>

<ol>
	<li>McNelis, J. C., Olefsky, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages,+immunity,+and+metabolic+disease.">Macrophages, immunity, and metabolic disease.</a> Immunity. 41 (1), 36-48 (2014).</li><li>Makki, K., Froguel, P., Wolowczuk, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+in+obesity-related+inflammation+and+insulin+resistance:+cells,+cytokines,+and+chemokines.">Adipose tissue in obesity-related inflammation and insulin resistance: cells, cytokines, and chemokines.</a> ISRN Inflamm. 2013, 139239 (2013).</li><li>DeFuria, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+promote+inflammation+in+obesity+and+type+2+diabetes+through+regulation+of+T-cell+function+and+an+inflammatory+cytokine+profile.">B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile.</a> Proc Natl Acad Sci U S A. 110 (13), 5133-5138 (2013).</li><li>Elgazar-Carmon, V., Rudich, A., Hadad, N., Levy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+transiently+infiltrate+intra-abdominal+fat+early+in+the+course+of+high-fat+feeding.">Neutrophils transiently infiltrate intra-abdominal fat early in the course of high-fat feeding.</a> J Lipid Res. 49 (9), 1894-1903 (2008).</li><li>Han, J. M., Levings, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+in+obesity-associated+adipose+inflammation.">Immune regulation in obesity-associated adipose inflammation.</a> J Immunol. 191 (2), 527-532 (2013).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+deficiency+and+pharmacological+stabilization+of+mast+cells+reduce+diet-induced+obesity+and+diabetes+in+mice.">Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice.</a> Nat Med. 15 (8), 940-945 (2009).</li><li>Nishimura, 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=CD8++effector+T+cells+contribute+to+macrophage+recruitment+and+adipose+tissue+inflammation+in+obesity.">CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity.</a> Nat Med. 15 (8), 914-920 (2009).</li><li>Koh, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+vascular+fraction+from+adipose+tissue+forms+profound+vascular+network+through+the+dynamic+reassembly+of+blood+endothelial+cells.">Stromal vascular fraction from adipose tissue forms profound vascular network through the dynamic reassembly of blood endothelial cells.</a> Arterioscler Thromb Vasc Biol. 31 (5), 1141-1150 (2011).</li><li>Peinado, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stromal-vascular+fraction+of+adipose+tissue+contributes+to+major+differences+between+subcutaneous+and+visceral+fat+depots.">The stromal-vascular fraction of adipose tissue contributes to major differences between subcutaneous and visceral fat depots.</a> Proteomics. 10 (18), 3356-3366 (2010).</li><li>Leggate, 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=Determination+of+inflammatory+and+prominent+proteomic+changes+in+plasma+and+adipose+tissue+after+high-intensity+intermittent+training+in+overweight+and+obese+males.">Determination of inflammatory and prominent proteomic changes in plasma and adipose tissue after high-intensity intermittent training in overweight and obese males.</a> Journal of Applied Physiology. 112 (8), 1353-1360 (2012).</li><li>Divoux, 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=Mast+cells+in+human+adipose+tissue:+link+with+morbid+obesity,+inflammatory+status,+and+diabetes.">Mast cells in human adipose tissue: link with morbid obesity, inflammatory status, and diabetes.</a> J Clin Endocrinol Metab. 97 (9), E1677-E1685 (2012).</li><li>Harford, K. A., Reynolds, C. M., McGillicuddy, F. C., Roche, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fats,+inflammation+and+insulin+resistance:+insights+to+the+role+of+macrophage+and+T-cell+accumulation+in+adipose+tissue.">Fats, inflammation and insulin resistance: insights to the role of macrophage and T-cell accumulation in adipose tissue.</a> Proc Nutr Soc. 70 (4), 408-417 (2011).</li><li>Lumeng, C. N., Bodzin, J. L., Saltiel, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+induces+a+phenotypic+switch+in+adipose+tissue+macrophage+polarization.">Obesity induces a phenotypic switch in adipose tissue macrophage polarization.</a> J Clin Invest. 117 (1), 175-184 (2007).</li><li>Morris, D. L., Singer, K., Lumeng, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+macrophages:+phenotypic+plasticity+and+diversity+in+lean+and+obese+states.">Adipose tissue macrophages: phenotypic plasticity and diversity in lean and obese states.</a> Curr Opin Clin Nutr Metab Care. 14 (4), 341-346 (2011).</li><li>Wentworth, J. 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=Pro-inflammatory+CD11c+CD206++adipose+tissue+macrophages+are+associated+with+insulin+resistance+in+human+obesity.">Pro-inflammatory CD11c+CD206+ adipose tissue macrophages are associated with insulin resistance in human obesity.</a> Diabetes. 59 (7), 1648-1656 (2010).</li><li>Wouters, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+classical+monocytes+are+associated+with+CD11c++macrophages+in+human+visceral+adipose+tissue.">Circulating classical monocytes are associated with CD11c+ macrophages in human visceral adipose tissue.</a> Sci Rep. 7, 42665 (2017).</li><li>Duan, 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=Distinct+macrophage+subpopulations+characterize+acute+infection+and+chronic+inflammatory+lung+disease.">Distinct macrophage subpopulations characterize acute infection and chronic inflammatory lung disease.</a> J Immunol. 189 (2), 946-955 (2012).</li><li>Li, F., 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=Autofluorescence+contributes+to+false-positive+intracellular+Foxp3+staining+in+macrophages:+a+lesson+learned+from+flow+cytometry.">Autofluorescence contributes to false-positive intracellular Foxp3 staining in macrophages: a lesson learned from flow cytometry.</a> J Immunol Methods. 386 (1-2), 101-107 (2012).</li></ol>

作者声明没有利益冲突。

这些方法描述如何分离 SVF 从增值税和 sAT 和量化的相对数量的免疫细胞在这些组织。此外, 这些方法还说明了如何确定特定单元格类型上标记的表达式。
组织免疫细胞的流式细胞术是一种有效的方法, 用于表型免疫状态的组织。组织免疫细胞的量化可以有许多应用。如结果所述, 有可能比较特定免疫细胞在患者组 (例如、瘦肉型与肥胖症) 之间的存在。此外, 通过对同一患者的血液进行流式细胞术, 可以研究循环细胞与组织细胞之间的关联。通过这个应用, 我们能够确定循环单核细胞的特定子集与亲炎性 CD11C+脂肪组织巨噬细胞16有关。
对所描述的协议的调整将扩展其应用, 因为许多可用的荧光抗体使流式细胞仪非常多才多艺。与不同的抗体几乎所有细胞类型可以区分和许多标记的表示可以被发现。此外, 还可以通过 permeabilizing 细胞膜来染色标记 intracellularly, 以允许荧光抗体的细胞内结合。这些特征允许不同的巨噬细胞种群以外的过度简化 M1 和 M2 巨噬细胞亚型。除了测量表面标记的表达, 蛋白质 (即, 细胞因子) 可以被染色 intracellularly 提供有关巨噬细胞功能的信息。此外, Ki67 等扩散标记用于量化扩散率。如所述, 巨噬细胞与 DCs 之间的区别是基于多边居民的 DC 标记水平。一般的巨噬细胞标记, 如 CD68 可以并入巨噬细胞面板 (1)。然而, CD68 需要染色 intracellularly 需要通透的细胞膜, 这是不可取的, 并将延长议定书。其他巨噬细胞标记是子集标记例如 CD163 和 CD206 或 CD11c, 后者被集成在这里提出的巨噬细胞小组。
在我们的外地管制小组中, 没有包括一个区分活细胞和死体细胞的标记, 这是可取的, 因为它允许比使用 FSC 和 SSC 更准确地排除死细胞。经常使用的是脱氧核糖核酸染色活性染料碘碘化物 (PI) 或 4 ', 6-diamidino-2-苯基吲哚 (DAPI) 并且自由胺反应染料例如活/死可修复的死细胞染色套件, 在不同的染料颜色可以使用。但是, 在修复单元格时不能使用 PI 和 DAPI。如《议定书》所述, 活/死可修复的红死细胞生存能力染色可以集成到两个面板中, 而不影响总的外地方案的门控策略。
此外, 数据表示为活细胞的百分比, 这意味着所有数据都是相对的。只有在流式细胞仪中输入确切的已知数目的单元格, 才能确定每个细胞类型的确切数字。在用计数室计算 SVF 分数中的细胞数后, 可以计算出细胞的近似数目。然而, 这个数字将不得不调整, 以用于隔离 SVF 的活检组织的数量, 但这有限制, 当比较瘦与肥胖。类似的肥胖人群中含有较少的脂肪细胞, 因为它们充满了脂质, 并大大扩大。这可能导致对免疫细胞数的低估, 如果提出作为免疫细胞的数量每克的在或每个脂肪。
在人类研究中, 纳入患者通常是在较长时间内进行标准化实验程序的重要意义。为比较患者之间的流式细胞仪数据, 有多种选择。如本协议所述, 在测量前可以对单元格进行固定, 以便在同一天对多个样本进行分析。这也可以通过冷冻 SVF 之前的染色, 这使得染色程序是平等的所有样本, 但细胞的生存能力可能受到影响。最后, 在本研究中, 还使用了荧光珠来安装补偿水平, 并采用了双周细胞仪跟踪珠来规范细胞仪的日常测量。这最后一个选择是最有效的测量样本从一项研究跨越了很长一段时间。
流式细胞术的限制因素一般是荧光的使用。可同时检测到的荧光标签数量因发射谱重叠而受到限制。然而, 通过智能化的流式仪表板开发和使用每个增值税或 sAT 样本的几个抗体鸡尾酒, 这个问题可以解决, 如本议定书所述。FMO 控制是资产管制系统面板开发的一个重要方面。通过使用该面板的所有抗体, 除了一个, 可以欣赏的潜在的自发荧光水平, 当比较 FMO 与整个面板。这样就可以精确地对人群进行浇口, 在设置新的 "流控流" 面板时应执行这些步骤。此外, 新一代的资产管制装置可以检测多达50个参数, 允许同时检测每个单元格的许多特性。另一个与荧光方面有关的问题是细胞的自体荧光, 特别是巨细胞。在细胞的励磁作用下激光 (主要与488毫微米波长励磁), 这些细胞发出一个荧光信号 (主要 < 640 毫微米) 可以重叠与抗体标签的发射光谱17,18。为此, 应测量无瑕细胞, 以确定每个通道中的自发荧光。有了这些知识, 荧光应该被选中, 显示超过 autofluorescent 信号的信号强度。在确定人群的门控策略时, 应考虑到 autofluorescent 的背景信号。因此, 通过本协议的应用和智能化的资产管制面板设计, 可以深入研究表型巨噬细胞子类型。新的不同在巨噬细胞和他们的作用可以描绘。

肥胖的特点是低品位的炎症1和浸润炎症免疫细胞在内脏和皮下 (增值税, sAT)。在增值税中积累的亲炎症免疫细胞导致胰岛素抵抗, 这是发展2型糖尿病的主要危险因素2。免疫细胞的先天和自适应免疫系统是发现在肥胖的, 如巨噬细胞, 肥大单元格, 中性白细胞, CD4+和 CD8+ T 细胞, B 细胞3,4,5,6 ,7。这些免疫细胞, 连同内皮细胞, 基质细胞, 脂肪胞祖, 成纤维细胞, 和毛细血管, 构成了 SVF8 , 是在9的促炎物质的主要来源。
通常由西方印迹10、qPCR11和免疫组化11等技术对 AT 的炎症状态进行调查。然而, 当使用这些技术时, 整个在, 脂肪细胞和 SVF, 使用。这使得很难确定在中存在的免疫细胞的数量和子集。免疫细胞有各种细胞标记来定义和分类它们, 如巨噬细胞。巨噬细胞在功能和细胞表面标记表达式12中显示显著的异质性。因此, 它们通常被归类为两个巨噬细胞群: M1 和 M2。M2 巨噬细胞通常被称为交替激活的巨噬细胞12,13并且居住在稀薄, 新陈代谢的正常人14。然而, 在肥胖期间, 一个表型开关发生从 M2 巨噬细胞到 M1 巨噬细胞。这些经典活化 M1 巨噬细胞表达 CD11C12和堆积在死脂肪体形成冠状结构13。它已经表明, CD11C+巨噬细胞在损害胰岛素的作用, 并与胰岛素抵抗在肥胖的人15。为了识别 M1 和 M2 巨噬细胞, 免疫组化是一种选择。该技术提供了有关组织中巨噬细胞位置的信息。但是, 它将限制可用于一个染色的标记的数量。此外, 也难以量化。因此, 为了研究不同的免疫细胞亚群在增值税和 sAT 存款, 我们开发了流式细胞术的方法。这种方法使我们有机会使用多个标记每个细胞与一个流式细胞分析, 以定义细胞子集和计数的每个子集存在的在存款。

<table>
	<tbody>
		<tr>
			<td>Centrifuge</td>
			<td>Hettich Rotanta 460R</td>
			<td>5660</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)-Ham&#8217;s F12</td>
			<td>Gibco ThermoFisher</td>
			<td>31330-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase XI from Clostridium histolyticum</td>
			<td>Sigma Aldrich</td>
			<td>C7657-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I from Clostridium histolyticum</td>
			<td>Sigma Aldrich</td>
			<td>C0130-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I from bovine pancreas</td>
			<td>Sigma Aldrich</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Merck</td>
			<td>1.01145.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumine</td>
			<td>Sigma Aldrich</td>
			<td>A4503-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Merck</td>
			<td>1.06688.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;M syringe Filcons</td>
			<td>BD Biosciences</td>
			<td>340613</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;M filter</td>
			<td>Greiner Bio-one</td>
			<td>542070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc block / human IgG</td>
			<td>Sigma Aldrich</td>
			<td>14506</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Merck</td>
			<td>1.04003.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-BV421</td>
			<td>Biolegend</td>
			<td>301324</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19-Fitc</td>
			<td>BD Biosciences</td>
			<td>555412</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-Fitc</td>
			<td>BD Biosciences</td>
			<td>561807</td>
			<td></td>
		</tr>
		<tr>
			<td>CD66b-Fitc</td>
			<td>BD Biosciences</td>
			<td>555724</td>
			<td></td>
		</tr>
		<tr>
			<td>CD56-Fitc</td>
			<td>BD Biosciences</td>
			<td>562794</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11c-APC-Cy7</td>
			<td>Biolegend</td>
			<td>337218</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>557748</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19-BV421</td>
			<td>Biolegend</td>
			<td>302234</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-V500</td>
			<td>BD Biosciences</td>
			<td>561416</td>
			<td></td>
		</tr>
		<tr>
			<td>CD56-APC</td>
			<td>Biolegend</td>
			<td>318310</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-PerCP-Cy5.5</td>
			<td>Biolegend</td>
			<td>300528</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8-APC-H7</td>
			<td>BD Biosciences</td>
			<td>641400</td>
			<td></td>
		</tr>
		<tr>
			<td>CD303-PE</td>
			<td>Biolegend</td>
			<td>354204</td>
			<td></td>
		</tr>
		<tr>
			<td>CD141-APC</td>
			<td>Biolegend</td>
			<td>344106</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS-Canto II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 v-shape well plate</td>
			<td>Greiner Bio-one</td>
			<td>651101</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS PH 7.4</td>
			<td>Gibco ThermoFisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tube 5 mL polystyrene round-bottom tube</td>
			<td>Corning</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG from human serum</td>
			<td>Sigma Aldrich</td>
			<td>I4506</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rat Ig, &#954;/Negative Control Compensation Particles Set</td>
			<td>BD Biosciences</td>
			<td>552844</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Ig, &#954;/Negative Control Compensation Particles Set</td>
			<td>BD Biosciences</td>
			<td>552843</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Red Dead Cell Stain Kit</td>
			<td>ThermoFisher</td>
			<td>L23102</td>
			<td></td>
		</tr>
		<tr>
			<td>IKA&#160;MS 3 basic shaker</td>
			<td>Sigma Aldrich</td>
			<td>Z645028-1EA</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of immune cells in human adipose tissue by using flow cytometry

在皮下和内脏脂肪组织中的免疫细胞浸润会导致低级别炎症, 导致肥胖相关并发症 (如2型糖尿病) 的发展。为了定量和定性地研究人体内的免疫细胞亚群, 我们开发了一种流式细胞仪方法。基质血管分数 (SVF), 含有免疫细胞, 从皮下和内脏在活检的胶原酶消化分离。脂肪细胞在离心后被除去。用流式细胞仪对 SVF 细胞进行染色, 筛选出多种膜结合标记, 以区分免疫细胞亚群和分析。由于这种方法, 可以检测和量化抗炎巨噬细胞亚群、树突状体细胞 (DCs)、B 细胞、CD4+和 CD8+ T 细胞以及 NK 细胞。此方法提供有关免疫细胞的详细信息, 以及每个特定子集的数量。由于有许多荧光抗体可用, 我们的流式细胞术方法可以调整, 以测量各种其他细胞和细胞内的标记感兴趣。

用流式细胞仪测量了 SVF 和 sAT 的分离。流式细胞仪测量根据细胞标记 (图 1A和1B) 生成显示不同细胞群的地块。首先, 通过绘制正散射宽度 (fsc-w) 和正散射区 (fsc A), 可以消除细胞团聚体的进一步分析, 将单个细胞作为低 FSC W。接下来, 选择活细胞, 并分别使用 FSC a 和侧散射区 (SSC a), 将正确大小和复杂度的细胞排除在细胞碎片之外。死细胞是小的, 因此可见作为一个独特的人口与一个小的 FSC-a。接下来, 使用泛白细胞标记 CD45 (面板1和 2,图 1A) 选择免疫细胞。为了分析巨噬细胞, 其他免疫细胞, 如 T 细胞 (CD3), B 细胞 (CD19), 中性粒细胞 (CD66b+ CD11b+) 和 NK 细胞 (CD56) 被排除在进一步的分析, 通过使用不同的抗体针对这些细胞, 但具有相同的fluorochrome。其余细胞的进一步细分是基于 CD11b 和 CD11c 表达。这导致以下人群: CD11b+ CD11c+巨噬细胞, CD11b+ CD11c-巨噬细胞, CD11b低/ CD11c+ DCs (1,图 1B)。平均荧光强度的测量允许量化 CD303 (浆 dc 标记) 和 CD141 (dc 标记) 的表达式, 在 CD11b+ CD11c+巨噬细胞和 CD11b低/ CD11c+ DCs 中。在 CD11b低/ CD11c+单元格中, 两个标记的表达式都较高, 确认 CD11b低/ CD11c+单元格为 dc (图 1C)。
CD45+单元格 (图 1A) 分别使用 CD3 和 CD19 划分为 T 细胞和 B 细胞。t 细胞被细分为 t 辅助细胞 (CD4+) 和细胞毒性 T 细胞 (CD8+)。最后, CD3-CD19 单元格被绘制成使用标记 CD56 (2,图 1D) 来量化 NK 细胞。每个门中的单元格数是量化的, 可用于计算所有活单元格类型的百分比 (表 3)。
可以计算每个科目的活细胞百分比, 允许计算一组中所有科目的平均值, 例如瘦或肥胖的男性, 显示特定免疫细胞的丰度,即, 赞成炎症的 CD11b+CD11c+内脏中的巨噬细胞 (图 2)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57319/57319fig1.jpg"> 图1。内脏脂肪组织的门控策略.(A) 具有正向散射宽度强度 (fsc W) 和正向散射区域强度 (fsc A) 的所有事件 (黑色) 的资产管制计划图包含一个只选择单个单元格 (红色) 的门, 后跟一个基于 FSC-a 和侧散射区域强度 (SSC a) 的外地跟踪图。包含选择活细胞 (浅绿色) 的门。下图与 SSC-A 和 CD45 荧光强度包含一个门选择所有 CD45+ (免疫) 细胞 (蓝色)。(B) CD19、CD3、CD66b 和 CD56 荧光强度与 CD11b 荧光强度相比较的图元, 以及选择所有 CD19、CD3、CD66b 和 CD56 阴性 (褐色) 的细胞以进一步分裂为人群的门。进一步细分的下一个情节基于 CD11b 和 CD11c 荧光强度。显示的闸门包含 CD11b+ CD11c+巨噬细胞 (深绿色)、CD11b+ CD11c-巨噬细胞 (紫色) 和 CD11b低/- CD11c+树突状细胞 (蓝色)。(C) 数量的 CD11b+、CD11c+或 CD11b低/- CD11c+单元格 (Y 轴), 它们显示了 CD303 和 CD141 的荧光强度 (X 轴) 的水平, 并相应地量化了平均值荧光强度 (多边基金会)。(D) 根据 CD3 和 CD19 荧光强度 (包括选择 T 细胞 (洋红)、B 细胞 (深绿色) 和非 autofluorescent 单元格 (绿色) 为两个 CD3 的 CD45, 显示以前的+填充 (蓝色) 的资产管制计划图和 CD19。下面的图是基于 CD4 和 CD8 荧光的, 选择 CD4+ (淡绿色) 和 CD8+ (洋红) T 单元格的门。皮下脂肪组织采用相同的门控策略。皮下脂肪组织采用相同的门控策略。此数字已从武泰et . 中进行了修改。16<a href="//cloudflare.jove.com/files/ftp_upload/57319/57319fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57319/57319fig2.jpg"> 图2。肥胖的增值税含有更多的促炎巨噬细胞.CD11b+ CD11c+巨噬细胞的数量显示为瘦身和肥胖男子增值税中所有活细胞的百分比。所有数据都是指电子扫描电镜;n = 20 为精益和 n = 31 为肥胖。**p ≤0.01 与精益。<a href="//cloudflare.jove.com/files/ftp_upload/57319/57319fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>目标</td>
			<td>定义目标</td>
			<td>呈现在</td>
			<td>Fluorochrome</td>
			<td>数量</td>
			<td>克隆</td>
		</tr>
<tr>
<td>CD11B</td>
			<td>髓素整合素标记</td>
			<td>粒细胞, 单核/巨噬, 树突状干细胞低和自然杀伤细胞</td>
			<td>BV421</td>
			<td>2.5 µL</td>
			<td>ICRF44</td>
		</tr>
<tr>
<td>CD19</td>
			<td>常见 B 细胞抗原</td>
			<td>b 细胞向 blastoid b 淋巴细胞和血浆 b 细胞发育的研究</td>
			<td>fitc</td>
			<td>3µL</td>
			<td>HIB19</td>
		</tr>
<tr>
<td>CD3</td>
			<td>普通 T 细胞抗原</td>
			<td>t 淋巴细胞, 自然杀伤细胞和胸腺</td>
			<td>fitc</td>
			<td>3µL</td>
			<td>UCHT1</td>
		</tr>
<tr>
<td>CD66B</td>
			<td>癌胚抗原 (CEA) 样糖蛋白家族成员</td>
			<td>粒</td>
			<td>fitc</td>
			<td>5µL</td>
			<td>G10F5</td>
		</tr>
<tr>
<td>CD56</td>
			<td>重糖化黏附蛋白</td>
			<td>自然杀伤细胞与自然杀伤细胞</td>
			<td>fitc</td>
			<td>5µL</td>
			<td>B159</td>
		</tr>
<tr>
<td>CD303</td>
			<td>II. 型跨膜糖蛋白</td>
			<td>浆树突状细胞</td>
			<td>体育</td>
			<td>1µL</td>
			<td>201A</td>
		</tr>
<tr>
<td>CD141</td>
			<td>血栓</td>
			<td>单核细胞/巨噬细胞低, 树突状细胞亚群</td>
			<td>apc</td>
			<td>1µL</td>
			<td>M80</td>
		</tr>
<tr>
<td>CD11C</td>
			<td>I 型跨膜糖蛋白;整合素αx</td>
			<td>单核/巨噬细胞, 树突状, 颗粒, 自然杀伤细胞, B 和 T 细胞的子集</td>
			<td>APC-Cy7</td>
			<td>0.5 µL</td>
			<td>Bu15</td>
		</tr>
<tr>
<td>CD45</td>
			<td>常见白细胞抗原</td>
			<td>所有人类白细胞包括淋巴细胞、单核细胞、粒粒、嗜酸性粒和胸腺</td>
			<td>PE-Cy7</td>
			<td>1µL</td>
			<td>HI30</td>
		</tr>
<tr>
<td>缓冲区</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>7.5 µl</td>
			<td>-</td>
		</tr>
</tbody></table>表1。用于anel 1 的抗体鸡尾酒, 用于识别巨噬细胞亚群和树突状体细胞种群. 所描述的抗体量用于分析一个样本。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>目标</td>
			<td>定义目标</td>
			<td>呈现在</td>
			<td>Fluorochrome</td>
			<td>数量</td>
			<td>克隆</td>
		</tr>
<tr>
<td>CD19</td>
			<td>常见 B 细胞抗原</td>
			<td>b 细胞向 blastoid b 淋巴细胞和血浆 b 细胞发育的研究</td>
			<td>BV421</td>
			<td>1µL</td>
			<td>HIB19</td>
		</tr>
<tr>
<td>CD3</td>
			<td>普通 T 细胞抗原</td>
			<td>t 淋巴细胞, 自然杀伤细胞和胸腺</td>
			<td>V500</td>
			<td>3µL</td>
			<td>UCHT1</td>
		</tr>
<tr>
<td>CD56</td>
			<td>重糖化黏附蛋白</td>
			<td>自然杀伤细胞与自然杀伤细胞</td>
			<td>apc</td>
			<td>5µL</td>
			<td>HCD56</td>
		</tr>
<tr>
<td>CD4</td>
			<td>I 型跨膜糖蛋白</td>
			<td>t 辅助细胞, 胸腺, 单核/巨噬, II. 型自然杀伤 T 细胞</td>
			<td>PerCP-Cy5。5</td>
			<td>1µL</td>
			<td>RPA-T4</td>
		</tr>
<tr>
<td>CD8</td>
			<td>二硫化物连接分子络合物的α亚基</td>
			<td>细胞毒性 T 细胞, 胸腺, 自然杀伤细胞子集</td>
			<td>APC-H7</td>
			<td>2µL</td>
			<td>SK1</td>
		</tr>
<tr>
<td>CD45</td>
			<td>常见白细胞抗原</td>
			<td>所有人类白细胞包括淋巴细胞、单核细胞、粒粒、嗜酸性粒和胸腺</td>
			<td>PE-Cy7</td>
			<td>1µL</td>
			<td>HI30</td>
		</tr>
<tr>
<td>缓冲区</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>10µl</td>
			<td>-</td>
		</tr>
</tbody></table>表2。用于流式检测的抗体鸡尾酒面板 2以确定 T 和 B 单元格的数量. 所描述的抗体量用于分析一个样本。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>巨噬细胞面板</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>门名</td>
			<td># 单元格</td>
			<td>% 的活</td>
		</tr>
<tr>
<td>单个单元格</td>
			<td>183054</td>
			<td></td>
		</tr>
<tr>
<td>生活</td>
			<td>10477</td>
			<td>100</td>
		</tr>
<tr>
<td>CD45</td>
			<td>4100</td>
			<td>39.13</td>
		</tr>
<tr>
<td>转储-
</td>
			<td>771</td>
			<td>7.36</td>
		</tr>
<tr>
<td>CD11C- CD11B+
</td>
			<td>430</td>
			<td>4。1</td>
		</tr>
<tr>
<td>CD11C+ CD11B+
</td>
			<td>104</td>
			<td>0.99</td>
		</tr>
<tr>
<td>CD11C+ CD11B低/
</td>
			<td>15</td>
			<td>0.14</td>
		</tr>
<tr>
<td>T 细胞, B 细胞, NK 细胞板</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>门名</td>
			<td>#Cells</td>
			<td>% 的活</td>
		</tr>
<tr>
<td>单个单元格</td>
			<td>34616</td>
			<td></td>
		</tr>
<tr>
<td>生活</td>
			<td>3728</td>
			<td>100</td>
		</tr>
<tr>
<td>CD45+
</td>
			<td>1589</td>
			<td>42.62</td>
		</tr>
<tr>
<td>NK 细胞</td>
			<td>29</td>
			<td>0.78</td>
		</tr>
<tr>
<td>CD3+
</td>
			<td>953</td>
			<td>25.56</td>
		</tr>
<tr>
<td>CD4+
</td>
			<td>601</td>
			<td>16.12</td>
		</tr>
<tr>
<td>CD8+
</td>
			<td>328</td>
			<td>8。8</td>
		</tr>
<tr>
<td>CD19+
</td>
			<td>20</td>
			<td>0.54</td>
		</tr>
</tbody></table>表3。不同细胞类型在增值税中的免疫细胞丰度。每个门中的单元格数, 以及基于生存单元总量的不同单元格类型的百分比。

Watch this Scientific Journal Video about Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry at JoVE.com

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

本文介绍了一种用流式细胞仪从脂肪组织中分离免疫细胞来分析脂肪组织免疫细胞含量的方法和后续分析。

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

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17.3K 次观看.  MUMC. 本文介绍了一种用流式细胞仪从脂肪组织中分离免疫细胞来分析脂肪组织免疫细胞含量的方法和后续分析。

视频: Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

科研

医学

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

白细胞亚群在暂时或永久的脑缺血模型体视学和流式细胞仪鉴定

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

人类的褐色脂肪组织的仓库的正电子发射断层扫描/计算机断层扫描自动分段及注册磁共振图像

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

应用 Videomicroscopy 评价人脂肪组织微血管功能

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

18F-fdg Petct 对活性人棕色脂肪组织的全身和区域定量

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

骨关节炎模型中骨髓、骨髓、淋巴结和合成组织内免疫细胞子集的流动细胞测量分析

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

使用附睾白色脂肪组织进行脂肪覆盖的胰岛移植

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this ...