Name: single-cell analysis of immunophenotype and cytokine production in peripheral whole blood via mass cytometry
Uploaded: 2018-06-26T13:30:00.000+00:00
Duration: 12 min 36 s
Description: Watch this Scientific Journal Video about Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry at JoVE.com

我们要感谢艾米 Pugh-伯纳德对她的智力投入和有益的评论。这项工作得到了 Boettcher 基金会韦伯-华林生物医学研究奖和 K23-1K23AR070897 从 NIH 到埃琳娜 W.Y. 谢的奖励数字的支持。她还得到了来自尤尼斯肯尼迪K12-HD000850 国家儿童健康和人类发展研究所以及露塞尔儿童健康基金会、斯坦福 CTSA UL1 TR001085 和儿童健康研究协会颁发的奖项编号的支持。斯坦福大学研究所。

这里描述的所有方法都已被科罗拉多大学的科罗拉多多机构审查委员会 (COMIRB) 批准。所有描述的程序应在无菌组织培养罩中执行, 除非另有说明, 过滤的吸管提示, 所有试剂过滤。
1. 外周全血处理试剂的制备
<ol><li>按照制造商的说明 (存储在-80 摄氏度), 在10毫米的 ruxolitinib 上, 根据生产商的指示, 对亚砜中的冻干试剂进行稀释, 准备整除数 (10–15 ul/小瓶)。在所有化验中保持亚砜浓度, 包括静态控制在0.2% 以下 (卷/卷)。</li>
	<li>在1微克/ul R848 (resiquimod) 库存整除数 (10–15 ul/小瓶), 稀释无菌水中的冻干试剂作为每个制造商的指示。存储在-80 摄氏度。</li>
	<li>在1微克/ul 中, 根据制造商的指示, 在无菌水中稀释冻干试剂, 制备脂多糖 (LPS) 库存整除数 (每瓶 5 ul 一次)。存储在-80 摄氏度。</li>
	<li>使用 PBS、0.5% BSA 和0.02% 南3制备细胞染色培养基 (CSM)。</li>
	<li>获取并保持准备蛋白转运抑制剂 (公共交通) 鸡尾酒 (材料表)。</li>
	<li>解冻和稀释 ruxolitinib 瓶 (10 毫米) 1:10 在无菌 PBS (1 毫米)。放置在室温下的组织培养罩。</li>
	<li>解冻和稀释 R848 瓶 (1 微克/µL) 1:10 在无菌 PBS (0.1 微克/µL)。放置在室温下的组织培养罩。</li>
	<li>解冻和稀释脂多糖瓶 (1 微克/µL) 1:100 在无菌 PBS (0.01 微克/µL)。放置在室温下的组织培养罩。</li>
	<li>在标准的37°c 水浴 (至少10分钟) 前温暖的无菌 RPMI (无 l-谷氨酰胺)。</li>
	<li>将溶解/固定缓冲器稀释 1:5, 过滤 ddH2O (工作浓度) 在台式上 (不需要无菌)。 注: 1 毫升的全血需要20毫升的工作浓度的溶解/固定缓冲。<ol>
<li>使溶解/固定缓冲器的5条件1毫升的全血每 (至少100毫升)。整除20毫升的工作浓度的溶解/固定缓冲器成50毫升圆锥管每毫升的全血 (保持溶解/修复解决方案在37°c, 直到需要)。对包含溶解/固定缓冲器的锥形管上的条件进行标记, 如下所示: T0 (时间零), T6 + 5R (时间6小时与 5 uM ruxolitinib), T6 + R848 (时间 6 h 以1微克/毫升 R848), T6 + LPS (时间 6 h 以0.1 微克/毫升 lps), T6 (时间 6 h)。</li>
	</ol>
</li>
	<li>标签圆底无菌聚苯乙烯管与帽和1毫升离心管与步骤1.10 相同的名称。</li>
</ol>2. 对周围全血的刺激和处理 (图 1)
<ol><li>将标记的圆底聚苯乙烯管从步骤 1.11 (T0、T6 + 5R、T6 + R848、T6 + LPS、T6) 放置在机架上。 注意: 除非另行指定, 否则在这种类型的管道中执行以下所有步骤。在组织培养罩和孵化器之间进行转移, 以维持不孕。</li>
	<li>添加1毫升37°c RPMI (无 l-谷氨酰胺) 的每一个管 (T0, T6 + 5R, T6 + R848, T6 + LPS, T6)。反转采血管几次, 每管加1毫升的全血, 和吸管上下几次混合彻底与 RPMI (稀释与 RPMI 防止在6小时潜伏期的血液聚集)。</li>
	<li>添加 10 ul 的 1:10 ruxolitinib 到 T6 + 5R。与 P1000 吸管彻底混合。将管架放在孵化器的37摄氏度, 开始定时孵化 (T = 0)。</li>
	<li>按照以下步骤处理 T0 示例。<ol>
<li>将 T0 管的全部内容注入一个含有20毫升37°c 工作浓度溶解/固定缓冲器的标记圆锥管。为优化细胞恢复, 用工作浓度溶解/固定缓冲液冲洗管。通过反转圆锥管混合。</li>
		<li>孵育在37°c 15 分钟, 以使溶解和固定。在固定后, 执行所有后续步骤在台式。在室温下, 离心细胞在 500 x g 处为5分钟。</li>
		<li>醒酒上清。并用重悬1毫升的冰冷 PBS 中的细胞分解颗粒, 然后用 PBS 填充圆锥到15毫升的体积。</li>
		<li>在室温下将细胞以 500 x g 为5分钟离心。醒酒上清。重复 PBS 洗涤 (步骤 2.4. 3–2.4 4) 如果小球是红色的。并用重悬1毫升内的细胞分解颗粒, 然后转移到标记的离心管 (步骤 1.11) 进行抗体染色。用一个10µL 的样本来计算使用自动单元计数器或 hemocytometer 的单元格。 注: 由于所有细胞都是固定的, 在这一点上, 没有必要包括生存的污点。</li>
		<li>在室温下将细胞以 500 x g 为5分钟离心。吸上清液, 将颗粒留在60µL 的残余容积中。把这个颗粒放在冰上, 直到其他条件的样品完成处理。</li>
	</ol>
</li>
	<li>在 T = 30 分钟, 将管架 (步骤 2.3) 移动到组织培养罩并执行以下操作。<ol>
<li>添加 10 ul 的0.1 微克/ul R848 到 T6 + R848。与 P1000 吸管彻底混合。</li>
		<li>添加 10 ul 的0.01 微克/ul lps 的 T6 + lps。与 P1000 吸管彻底混合。</li>
		<li>添加 4 ul 的蛋白转运抑制剂 (股票在 500X) 到 T6, T6 + 5R, 和 T6 + LPS (但不是T6 + R848), 并返回到孵化器的样品, 直到 T = 6 h. 每2小时与一个 P1000 吸管彻底混合。 注: R848 是一种内质 TLR 激动剂, 因此需要在增加蛋白转运抑制剂的时间延迟, 以允许进入其目标受体。</li>
	</ol>
</li>
	<li>在 T = 2 h, 添加 4 ul 的蛋白转运抑制剂鸡尾酒 (股票在 500X) 到 T6 + R848 管。将所有样品与 P1000 吸管混合, 并将机架返回到孵化箱, 直到 T = 6 小时。</li>
	<li>将样品与 P1000 再混合一次, 在 T = 4 小时。</li>
	<li>在 T = 6 h, 处理 T6, T6 + LPS, T6 + R848, T6 + 5R 血液样品管如步骤2.4 中所述 T0 样品。</li>
	<li>将细胞小球储存在-80 °c 的残余 CSM 体积内, 以处理未来的日期。或者, 进行条形码和抗体染色而不储存。</li>
</ol>3. 裂解/固定血细胞条码
<ol><li>在冰上慢慢解冻裂解/固定细胞样品从-80 °c 存贮;最多有20个样本可以用独特的条形码标记, 并使用这个系统进行汇集。用 PBS 稀释10X 条形码烫发缓冲器 1:10;使足够的缓冲区为每样3毫升。</li>
	<li>用 CSM 和1X 条形码烫发器填充一个无菌槽。加入1毫升的冰冷 CSM 到新鲜解冻的样品, 与吸管彻底混合, 并转移到各自预先标记的聚丙烯集束管。</li>
	<li>用一个 10 ul 样本来计数细胞使用自动的细胞计数器或 hemocytometer。将单元格计数规范化为每个簇管中每个样本的 1.5–2 x 106个单元格: 移除并丢弃超过 2 x 106个细胞的多余细胞的体积。</li>
	<li>在室温下将细胞以 600 x g 为5分钟离心。并用重悬在1毫升1X 条形码烫发缓冲细胞与多通道吸管和离心机在 600 x g 5 分钟室温。从上清中吸取。</li>
	<li>按条形码键上指示的顺序将机架上的簇管排成一行, 以便示例与条形码匹配。添加 800 ul 1X 条形码烫发缓冲通过多通道吸管到所有样本的集束管没有接触的细胞颗粒与吸管提示 (不混合), 以减少细胞损失。用集束管把架子搁在一边。</li>
	<li>去除20丛 Pd 条形码试剂盒从-20 °c 和解冻在室温下。添加 100 ul 1X 条形码烫发缓冲器, 彻底混合, 并转移 120 ul 的悬浮条码混合到相应的细胞样本在集群管。</li>
	<li>通过多通道吸管彻底混合, 使单独条形码样品之间没有交叉污染。在室温下孵化出30分钟的聚束管, 允许条形码对细胞进行标记。</li>
	<li>离心机在室温下为 600 x 克, 5 分钟。吸入上清, 然后并用重悬1毫升的 CSM。</li>
	<li>离心机和并用重悬在 CSM 再次像在步骤3.8。 注意: 每个示例现在都用一个唯一的条形码进行标记, 并且样品可以被汇集。</li>
	<li>离心机在室温下 600 x 克, 5 分钟, 吸上清液。用单吸管和使用相同的尖端, 转移所有细胞颗粒在〜 70–80 ul 残余体积到一个聚苯乙烯管。不要弹出吸管尖端;用这个尖端把单吸管放在一边。</li>
	<li>用多声道吸管和新的提示, 添加〜 100 ul CSM 每一个原始的集束管, 以最大限度地恢复细胞。用单吸管与提示预留, 转移所有细胞颗粒在 100 ul 剩余体积到同一聚苯乙烯管。</li>
	<li>添加 CSM 到顶部的聚苯乙烯管 (~ 3 毫升)。计数并记录池内条形码集的单元格号。离心机在室温下 600 x 克, 5 分钟, 吸上清液。在同一天对条形码样品进行染色。 注意: 在条形码过程中期望20–30% 细胞丢失是正常的。</li>
</ol>4. 条形码裂解/固定血细胞染色及质量细胞仪分析的制备
注: 染色抗体的每1X 滴度 (1 ul 抗体每 100 ul 染色反应), 通常可以染色 3–4 x 106细胞。因此, 当所有的条形码样本被汇集成一个管, 抗体的数量必须扩大。如果20条形码样品共计 30 x 106个细胞, 并且每1X 滴度能染色 3–4 x 106个细胞, 条形码样品只需要10X 滴度, 相反单独地染色每个样品, 需要20X 抗体 (1X 每单独管)。抗体浓度的细胞数应仔细滴定每一个抗体鸡尾酒 (这里没有讨论)。
<ol><li>用抗体 (材料表) 染色细胞, 用于表面染色和细胞因子诱导。<ol>
<li>通过计算要添加的量和吹打错误 (例如, 如果在每个样品中染色 2 x 106个细胞的20条形码样本, 就需要进行表面染色鸡尾酒; 对于最终的卷计算,通过制作10.5X 最终染色液来补偿吹打误差)。</li>
		<li>添加10X 的表面染色量的条形码细胞颗粒。为了减少细胞损失, 用同样的尖端, 混合和测量表面污渍和细胞颗粒的体积。</li>
		<li>根据细胞体积和染色鸡尾酒, 增加 CSM 到最终染色量 50 ul 每数量的细胞样本 (即, 如果运行一组20样品, 50 ul X 20 = 1 毫升)。孵育30分钟, 在4摄氏度。每15分钟搅动样品以促进均匀染色。</li>
		<li>在表面染色孵化过程中, 用过滤后的 ddH2的稀释 1:10, 准备烫发/洗涤缓冲器 (材料表), 准备通透2毫升的容积, 以及5毫升的洗涤。保持在4摄氏度或冰上。</li>
		<li>顶部的表面染色管与 CSM, 和离心机在 600 x g 在4°c 5 分钟。并用重悬条形码样品在2毫升4°c, 1:10 稀释烫发/洗涤缓冲器。孵育在4°c 20–30分钟完全 permeabilize 细胞。</li>
		<li>离心机在 600 x g 在4°c 5 分钟和吸入上清。</li>
		<li>对于细胞内染色, 遵循类似的染色步骤 (如表面染色)。然而, 使用4°c, 1:10 稀释烫发/洗涤缓冲, 而不是 CSM, 使总染色量, 使细胞保持在一个 permeabilizing 的环境中整个细胞内染色。</li>
		<li>添加细胞内抗体鸡尾酒的表面染色和透细胞颗粒 (步骤 4.1. 2–4.1 17)。将总染色量的 50 ul 每数量的细胞样本。孵育60分钟, 在4摄氏度。每15分钟搅动样品, 以确保均匀染色。</li>
		<li>在细胞内染色时, 准备 intercalator 溶液: 900 ul 的过滤 PBS + 100 ul 的过滤16% 粉煤灰 (最终浓度1.6% 粉煤灰) + 0.2 ul 的 500 uM 的 intercalator 染料 (材料表)。</li>
		<li>顶出细胞颗粒 + 细胞内抗体鸡尾酒与冷1:10 烫发/洗涤缓冲。</li>
		<li>离心机在 600 x g 在4°c 5 分钟, 并吸入上清。顶部的染色管与 CSM。计数并记录单元格号。离心机在 600 x g 在4°c 5 分钟, 并吸入上清。从步骤4.9 并用重悬1毫升的 intercalator 溶液中的细胞。在室温下孵育至少20分钟以充分插层, 或隔夜在4摄氏度 (intercalator 溶液中的样品可以停留在4摄氏度, 直到1周, 然后再在质量细胞仪上运行它们)。</li>
	</ol>
</li>
	<li>为大众细胞术仪器准备细胞。<ol>
<li>顶出管与3毫升过滤 ddH2O, 和离心机在 600 x g 5 分钟在4°c。并用重悬在3毫升的过滤 ddH2o 的细胞通过这个悬浮通过100微米的过滤器, 以消除任何碎片或聚集可能会堵塞的大规模细胞术的仪器。</li>
		<li>计数并记录过滤后的细胞数。离心机在 600 x g 5 分钟在4°c</li>
	</ol>
</li>
	<li>通过稀释1:10 在过滤 ddH2O 准备定标珠解答 (材料表)。</li>
	<li>并用重悬染色细胞在所需体积的1:10 稀释校准珠溶液, 以达到细胞浓度为 ~ 1 x 106细胞/毫升。 注: 对于 CyTOF1 在45µL/分钟, 推荐的最佳是 5 x 105细胞/毫升;为太阳神在30µL/分钟, 7.5 x 105细胞/毫升。</li>
	<li>继续运行该样本的大规模细胞术仪器9。</li>
</ol>

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作者没有什么可透露的。

在这里, 我们描述了一个新的, 单细胞, 蛋白质组学方法, 同时评估多种免疫细胞类型和检测其各种细胞因子扰动的环境中的患者特异性 "致病性" 外周血血浆循环因素。该方法采用外周全血作为分析载体, 以质细胞仪作为评价免疫细胞表型和功能异常的工具。该方法易于应用于人体和小鼠的研究21, 并与其他免疫功能分析 (如检测信号异常14) 兼容。
<p class="jove_co...

自身免疫性疾病是影响3-8% 人口发病率和死亡率的主要原因。在美国, 自体免疫性疾病是青年和中年妇女死亡的主要原因 (年龄 < 65 岁)1,2。自身免疫性疾病的特点是异质临床表现和多种基础免疫学过程。非均质谱在不同的疾病中有很好的表现, 如联合参与类风湿关节炎 (RA) 和多发性硬化症 (MS) 的神经系统疾病。然而, 这一水平的异质性也表现在单一的紊乱, 如系统性红斑狼疮 (SLE): 一些病人可能出现肾脏病理, 而其他人患有血液或联合介入3。
自身免疫性疾病的潜在发病反映了临床异质性, 包括多先天和自适应免疫细胞亚群的自动和超活化, 以及伴随失调细胞因子的产生。虽然细胞因子在自身免疫性疾病的发病机制中起着举足轻重的作用, 但了解其在疾病机理中的具体作用已证明是具有挑战性的。细胞因子的特点是多效性 (一个细胞因子可以对不同的细胞类型有多种影响), 冗余 (多个细胞因子可以有相同的效果), 二元性 (一个细胞因子可以有亲或抗炎作用在不同的条件下),和可塑性 (细胞因子可以塑造成一个角色不同于它的 "原物" 一, 取决于环境)4,5,6。因此, 人口水平的方法不能区分异质细胞对同一 "细胞因子环境" 的反应。同样, 研究的设计, 专注于免疫系统的一个特定方面 (单一细胞类型或细胞因子), 不提供全球评估所有的因素涉及复杂的自身免疫性疾病。虽然以血清为基础的研究为自身免疫提供了重要的洞察力, 但它们并没有给出失调细胞因子的特定细胞来源。
最近, 我们开发了单细胞蛋白质组学方法, 同时评估多种免疫细胞类型, 并检测其在特定的 "致病性" 外周血血浆循环因子的环境中的各种细胞因子扰动。这里描述的工作流的特点是使用完整的周围全血样本, 而不是孤立的外周血单个核细胞 (PBMCs)。外周全血代表最生理相关的车辆, 以研究系统性免疫介导的疾病, 包括 1) 非单核血细胞经常参与自身免疫性疾病 (即中性粒细胞, 血小板) 和 2) 血浆诸如核酸、免疫复合物和细胞因子等循环因子, 具有免疫激活作用。为了捕捉 "内在致病性" 失调细胞因子的产生, 外周血标本立即处理后, 血液提取 (T0, 时间零), 并在6小时的孵化后37°c (生理体温) 与蛋白质运输抑制剂在没有任何外源刺激条件 (T6, 时间6小时), 以检测细胞因子的生产 (积累, T6-T0), 这将反映 "内在" 疾病状态 (图 1)。为了研究反应信号通路在与疾病相关的免疫应答中的失调过程, 外周血样本被治疗 (6 小时孵化37°c 与蛋白质转运抑制剂) 与外源刺激的条件, 反映疾病的发病机制, 例如, 在 SLE (T6 + R848, 时间6小时与1µg/毫升 R848) 的情况下类似的像样受体 (TLR) 激动剂, 以检测细胞因子的产生, 反映对核酸的反应 (比较 T0 和 T6 vs T6 +R848,图 1)。为了研究可用疗法的免疫调节作用, 因为它们与特定患者的精确免疫失调过程有关, 所以在相关治疗中用 JAK 抑制剂治疗外周血标本。集中 (这里, 5 uM ruxolitinib;T6 + 5R, 时间6小时与 5 uM ruxolitinib), 以检测 "内在" 疾病状态的变化反应的药物 (T0 vs T6 vs T6 + 5R,图 1)。为这项研究选择了 JAK 抑制剂, 因为 JAK 抑制剂已证明是成功的治疗自身免疫性疾病, 如 RA。
为了同时评价上述在多免疫细胞亚群中的失调过程, 对 SLE 患者的外周血样本和健康控制进行了处理, 并通过质细胞术进行了分析。质细胞术, 也称为细胞术-飞行时间, 提供了40多个参数的单细胞分析, 没有光谱重叠7,8,9的问题。这种技术利用稀土金属同位素的形式, 可溶性金属离子作为标签绑定到抗体, 而不是显影10。关于质量细胞术技术平台的其他细节 (即调谐和校准, 样品采集) 可以在 Leipold 等和麦卡锡等中找到 。11,12高维度的大规模细胞术能够同时测量的多种细胞因子整个先天和自适应免疫细胞亚群与单细胞粒度 (材料表)。
目前的常规临床和实验室参数往往不敏感或具体到足以检测正在进行的疾病活动或对特定的免疫调节剂13的反应, 反映了需要更好地描绘潜在的免疫驱动 flare 的更改。鉴于细胞因子失调在自身免疫性疾病中的普遍存在, 大量的治疗方法使用的抗体或小分子抑制剂靶向细胞因子或信号蛋白参与调控细胞因子的生产有最近出现。在它的基本格式, 这里描述的外周血分析方法提供了一个平台, 以确定病人特定的失调细胞亚群和他们的异常细胞因子产生的自身免疫性疾病的系统性表现。这种方法允许个性化的治疗选择, 因为具体的失调细胞因子可以确定, 和特定的治疗方案, 可以测试在体内, 以评估他们的能力 immunomodulate 病人特定的疾病过程。

<table><tbody><tr><td>Ruxolitinib</td><td>Santa Cruz</td><td>SC-364729A</td><td>Stock Conc: 10 mM; Final Conc: 5 &amp;mu;M</td></tr><tr><td>R848</td><td>Invivogen</td><td>tlrl-r848-5</td><td>Stock Conc: 1 &amp;mu;g/&amp;mu;L; Final Conc: 1 &amp;mu;g/mL</td></tr><tr><td>LPS-EK</td><td>Invivogen</td><td>tlrl-eklps</td><td>Stock Conc: 1 &amp;mu;g/&amp;mu;L; Final Conc: 0.1 &amp;mu;g/mL</td></tr><tr><td>Sterile PBS</td><td>Lonza</td><td>17-516F</td><td></td></tr><tr><td>Lyse/Fix Buffer</td><td>BD biosciences</td><td>558049</td><td>Stock Conc: 5X; Final Conc: 1X (dilute in ddH2O)</td></tr><tr><td>BD Phosflow perm/wash buffer I</td><td>BD biosciences</td><td>557885</td><td>Stock Conc: 10X; Final Conc: 1:10 (dilute in ddH2O)</td></tr><tr><td>RPMI</td><td>Gibco</td><td>21870-076</td><td></td></tr><tr><td>Sodium Azide (NaN3)</td><td>Sigma-Aldrich</td><td>S-8032</td><td>Stock Conc: &gt;99.9%; Final Conc: 0.0002</td></tr><tr><td>Protein Transport Inhibitor (PTI)</td><td>eBiosciences</td><td>00-4980-93</td><td>Stock Conc: 500X; Final Conc: 1X</td></tr><tr><td>DNA Intercalator</td><td>Fluidigm</td><td>201192B</td><td>Stock Conc: 500 &amp;mu;M; Final Conc: 0.1 &amp;mu;M</td></tr><tr><td>Cell Staining Media (CSM)</td><td></td><td></td><td>PBS + 0.5% BSA, 0.02% NaN3</td></tr><tr><td>MaxPar Barcode Perm Buffer</td><td>Fluidigm</td><td>201057</td><td>Stock Conc: 10X; Final Conc: 1X</td></tr><tr><td>20-plex Pd Barcode Set</td><td>Fluidigm</td><td>S0014</td><td>Stock Conc: n/a; Final Conc: n/a</td></tr><tr><td>EQ &lt;sup&gt;TM&lt;/sup&gt; Four Element Calibration Beads</td><td>Fluidigm</td><td>201078</td><td>Stock Conc: 10X; Final Conc: 1X</td></tr><tr><td>16% MeOH-free Formaldehyde Solution</td><td>Thermo</td><td>28908</td><td>Stock Conc: 16% (w/v); Final Conc: 1.6% (w/v)</td></tr><tr><td>Sterile round bottom polystyrene tubes</td><td>VWR</td><td>60818-496</td><td>Stock Conc: n/a; Final Conc: n/a</td></tr><tr><td>Polypropylene cluster tubes</td><td>Light Labs</td><td>A-9001</td><td>Stock Conc: n/a; Final Conc: n/a</td></tr><tr><td>Helios CyTOF instrument</td><td>Fluidigm</td><td>Helios</td><td>All solutions to be used in CyTOF analysis need to be free of metal contamination. ddH2O is used in the preparation of any solutions should have a resistivity of at least 18.0 M&amp;Omega;.cm. ddH2O and any self-prepared solutions should be stored in new plastic or glass bottles that have never been autoclaved.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies used for Mass Cytometry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Surface markers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD1c</td><td>Biolegend</td><td>L161</td><td>Mass: 161</td></tr><tr><td>CD3</td><td>BD</td><td>UCHT1</td><td>Mass: 144</td></tr><tr><td>CD4</td><td>Biolegend</td><td>SK3</td><td>Mass: 174</td></tr><tr><td>CD7</td><td>BD</td><td>M-T701</td><td>Mass: 149</td></tr><tr><td>CD8</td><td>Biolegend</td><td>SK1</td><td>Mass: 142</td></tr><tr><td>CD11b</td><td>Fluidigm</td><td>ICRF44</td><td>Mass: 209</td></tr><tr><td>CD11c</td><td>BD</td><td>B-ly6</td><td>Mass: 152</td></tr><tr><td>CD15</td><td>BD</td><td>HI98</td><td>Mass: 115</td></tr><tr><td>CD14</td><td>Biolegend</td><td>M5E2</td><td>Mass: 154</td></tr><tr><td>CD16</td><td>eBioscience/Thermo</td><td>B73.1</td><td>Mass: 165</td></tr><tr><td>CD19</td><td>Santa Cruz</td><td>SJ25C1</td><td>Mass: 163</td></tr><tr><td>CD21</td><td>Biolegend</td><td>Bu32</td><td>Mass: 141</td></tr><tr><td>CD27</td><td>BD</td><td>L128</td><td>Mass: 155</td></tr><tr><td>CD38</td><td>Fluidigm</td><td>HIT2</td><td>Mass: 172</td></tr><tr><td>CD45 total</td><td>Biolegend</td><td>HI30</td><td>Mass: 89</td></tr><tr><td>CD45RA</td><td>Biolegend</td><td>HI100</td><td>Mass: 153</td></tr><tr><td>CD56</td><td>Miltenyi</td><td>REA196</td><td>Mass: 168</td></tr><tr><td>CD66</td><td>BD</td><td>B1.1/CD66</td><td>Mass: 113</td></tr><tr><td>CD86</td><td>Fluidigm</td><td>IT2.2</td><td>Mass: 150</td></tr><tr><td>CD123</td><td>Fluidigm</td><td>6H6</td><td>Mass: 143</td></tr><tr><td>CD278/ICOS</td><td>Biolegend</td><td>C398.4A</td><td>Mass: 156</td></tr><tr><td>CD179/PD1</td><td>Biolegend</td><td>EH12.2H7</td><td>Mass: 162</td></tr><tr><td>IgD</td><td>Biolegend</td><td>IA6-2</td><td>Mass: 146</td></tr><tr><td>IgM</td><td>Biolegend</td><td>MHM-88</td><td>Mass: 151</td></tr><tr><td>CXCR5</td><td>BD</td><td>RF8B2</td><td>Mass: 173</td></tr><tr><td>HLADR</td><td>Biolegend</td><td>L243</td><td>Mass: 167</td></tr><tr><td>&lt;strong&gt;Cytokines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>IL-1&amp;alpha;</td><td>Biolegend</td><td>364-3B3-14</td><td>Mass: 147</td></tr><tr><td>IL-1&amp;beta;</td><td>Biolegend&amp;nbsp;</td><td>H1b-98</td><td>Mass: 169</td></tr><tr><td>IL-1RA</td><td>Santa Cruz&amp;nbsp;</td><td>AS17</td><td>Mass: 157</td></tr><tr><td>IL-6</td><td>Biolegend</td><td>MQ2-13A5</td><td>Mass: 164</td></tr><tr><td>IL-8</td><td>BD</td><td>E8N1</td><td>Mass: 160</td></tr><tr><td>IL-12/IL-23p40</td><td>Biolegend&amp;nbsp;</td><td>C8.6</td><td>Mass: 171</td></tr><tr><td>IL-17A</td><td>Biolegend&amp;nbsp;</td><td>BL168</td><td>Mass: 148</td></tr><tr><td>IL23p19</td><td>eBioscience/Thermo</td><td>23dcdp</td><td>Mass: 176</td></tr><tr><td>MIP1&amp;beta;</td><td>BD</td><td>D21-1351</td><td>Mass: 158</td></tr><tr><td>MCP1</td><td>BD</td><td>5D3-F7</td><td>Mass: 170</td></tr><tr><td>IFN&amp;alpha;</td><td>Miltenyi&amp;nbsp;</td><td>LT27:295</td><td>Mass: 175</td></tr><tr><td>IFN&amp;gamma;</td><td>Biolegend</td><td>4S.B3</td><td>Mass: 165</td></tr><tr><td>PTEN</td><td>BD</td><td>A2B1</td><td>Mass: 159</td></tr><tr><td>TNF&amp;alpha;</td><td>Biolegend</td><td>Mab11</td><td>Mass: 166</td></tr><tr><td>&lt;strong&gt;Note&lt;/strong&gt;: If the manufacturer is stated as Fluidigm, this antibody was purchased from Fluidigm with metal pre-conjugated. If the manufacturer is stated as other than Fluidigm, this antibody was self-conjugated using the MaxPar Multi-Metal Labeling&amp;nbsp; Kit (Fluidigm Cat: 201300) according to manufacturer protocol.&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

single-cell analysis of immunophenotype and cytokine production in peripheral whole blood via mass cytometry

细胞因子在自身免疫性疾病的发病机制中起着举足轻重的作用。因此, 对细胞因子水平的测量一直是多项研究的重点, 试图了解导致自我耐受和随后的自身免疫性崩溃的精确机制。迄今为止的方法都是基于对免疫系统的一个特定方面 (单个或少数细胞类型或细胞因子) 的研究, 并且不提供对复杂自身免疫性疾病的全球评估。虽然以血清为基础的研究为自身免疫提供了重要的洞察力, 但它们并没有给出失调细胞因子的特定细胞来源。本文介绍了在 "固有" 患者特定血浆循环因子的背景下, 综合评价多种免疫细胞亚群细胞因子生成的综合性单细胞方法。这种方法可以监测患者特定的免疫表型 (表面标记) 和功能 (细胞因子), 无论是在其本机 "固有致病性" 疾病状态, 或在存在的治疗药物 (在体内或体内).

图 1展示了用于刺激和处理外周血液样本的工作流, 包括血液样本整除数的分配、添加刺激剂的时间、蛋白质转运抑制剂鸡尾酒和孵化时间直到红细胞 (红细胞) 溶解和固定。刺激剂的选择取决于要评估的信号和细胞因子通路。例如, 在这里描述的协议中, TLR 激动剂被用来评估不同细胞类型的先天免疫传感机制。选择了代表性细胞外 (LPS, TLR4 激动剂) 和?...

Watch this Scientific Journal Video about Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry at JoVE.com

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

在这里, 我们描述了一个单细胞蛋白质组学方法, 以评估免疫表型和功能 (细胞因子诱导) 改变周围全血标本, 分析通过大规模细胞术。

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

single-cell-analysis-immunophenotype-cytokine-production-peripheral

Research

JoVE Journal

Immunology and Infection

9.4K 次观看.  University of Colorado School of Medicine. 在这里, 我们描述了一个单细胞蛋白质组学方法, 以评估免疫表型和功能 (细胞因子诱导) 改变周围全血标本, 分析通过大规模细胞术。

视频: Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

科研

生物工程

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

枚举主要外周血白细胞人口多中心临床试验，使用全血表型分析

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

免疫与感染

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

双氟铬流式细胞仪对七种免疫细胞子集的判别

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

<meta charset="utf8"></head><body>用于分析真菌和病毒免疫的优化全血免疫检测

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 &#181;L) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.

<meta charset="utf8"></head><body>作者聚焦:使用全血检测推进重症监护患者的免疫监测

Whole blood-based immunoassays provide a facile and resource-efficient tool to analyze antigen-specific immunity for diagnostic and research purposes. This article provides an optimized whole blood-based protocol with dual co-stimulation for comprehensive analysis of host immunity to fungal and viral pathogens, including a low-volume version for pediatric patients and small animals.

具有双重共刺激的全血测定，用于宿主对真菌和病毒病原体免疫的抗原特异性分析

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

摘要

探索更多视频

此视频中的章节

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

枚举主要外周血白细胞人口多中心临床试验，使用全血表型分析

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

全血流式细胞术分析人单核细胞亚群的表征

双氟铬流式细胞仪对七种免疫细胞子集的判别

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

具有双重共刺激的全血测定，用于宿主对真菌和病毒病原体免疫的抗原特异性分析

具有双重共刺激的全血测定，用于宿主对真菌和病毒病原体免疫的抗原特异性分析

具有双重共刺激的全血测定，用于宿主对真菌和病毒病原体免疫的抗原特异性分析

样品制备质谱术分析