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。
用户应该认识到一些可能影响此过程成功的变量。根据我们的经验, 应在2小时内处理血液样本, 以避免细胞内细胞因子的基线诱导 (未显示数据)。血液样本应保持室温 (25 °c), 同时等待处理。血液样本可以收集在肝素钠或 EDTA 管, 不干预的描述化验。应用顺铂22进行大容量细胞术, 可评估活细胞和死体细胞的鉴别。但是, 上述评估在上文所述的议定书中省略, 假定血液在收集后立即处理 (或在2小时以内)。此外, 即使样品在加工过程中被延迟, 由于缺乏冷冻保存, 也不能忽略活/死的歧视。不过, 我们建议使用顺铂进行生存性歧视, 以进行任何研究, 其中细胞死亡可能影响下游分析结果。
此外, 我们发现, 对刺激剂的仔细选择和滴定是评估靶向免疫通路/细胞因子诱导的必要条件。首先, 刺激剂的选择应基于 1) 的免疫通路, 目的是进行/评估, 2) 目标功能读数 (无论是信号蛋白或细胞因子生产)。例如, 如果目标是评估 t 细胞活化和不同的 t 辅助细胞 (Th) 亚群及其各自的细胞因子诱导, PMAIONO 或组合 anti-CD3/anti-CD28 将是一个适当的刺激条件。但是, 如果目标是评估单核细胞子集激活, TLR 激动剂 (或两者的组合) 可能是最好的。其次, 这些刺激剂的浓度需要优化, 以诱发细胞活化和细胞因子诱导, 而不严重改变细胞表面标记物。使用太少的刺激剂不会导致所需的细胞类型的活化, 也不会观察到细胞因子的诱导, 而过多的使用会导致细胞过多死亡, 免疫细胞表面标记物的变化, 从而使种群识别在静态条件下不会出现在受激条件下。第三, 刺激的动力学也是一个重要的变量。虽然 6 h 以前被发布了作为最佳的刺激时间期限捕获促进炎症细胞因子的最大诱导在反应 TLR 刺激14,23, 可能需要不同的刺激 timepoint其他特工。同样, 使用任何治疗药物体外与外周血标本也应与刺激剂相同的方式滴定, 同时密切关注浓度和 timepoint。
在本议定书中, 我们使用新鲜的外周全血样本, 因为细胞内细胞因子诱导 (和检测) 是比较健壮和有效的与冷冻保存样品相比。我们在这里描述了使用全血, 而不是 PBMCs, 因为血浆循环因素的存在导致了与疾病过程有关的免疫异常的 "内在" 性质。这些血浆循环因素可能会干扰附加到周围全血的刺激条件, 例如添加抗 IgM 和抗 IgG 以接触 B 细胞受体, 因为它们受血浆循环抗体和红细胞的约束。因此, 在使用外周全血时, 仔细选择刺激条件对成功的实验阅读是至关重要的。使用外周全血有其优点 (在体内平台) 和缺点 (干扰血浆循环因素)。然而, PBMCs 提供了一组不同的 "利弊"。当新鲜的 PBMCs (而不是全血) 经历相同的过程/协议, 并与刺激使用不同的 TLR 剂, 我们观察到相同的 "模式" 的细胞因子诱导, 虽然轻微小于14级,23。根据这一协议, 使用冷冻 PBMCs 导致一个不太健壮的细胞因子诱导, 因此我们建议不要直接比较的结果与冷冻保存后处理新鲜的样品。
鉴于新鲜的和冰冻的样品的警告, 以及更倾向于使用新鲜的样品以获得最佳的数据质量, 这里描述的协议适用于前瞻性的人类研究, 在这个时间段内收集病人样本。外周血标本可按描述进行处理, 一旦达到红细胞溶解和固定阶段, 样品可储存在-80 摄氏度, 以后分批染色。这种技术方法, 虽然有利于大多数人类研究, 也提出了挑战, 使用抗体, 可以绑定到目标表位后固定。材料表列出了各自抗体的克隆, 并对其进行了检测并证明了特定的染色后固定。多样品的条形码 (n = 20) 提供了1的优点) 减少了技术的变异性, 因为多个样本被一起在一管中染色, 因此使样品的比较一致和可靠;2) 减少总抗体的使用 (见议定书第4.1 节);和 3) "6 不同钯同位素/选择 3" 的条形码方案导致双峰 (细胞阳性超过4钯同位素) 的排除。这些优势在 Zunder 和菲克等方面进行了详细的讨论。24另外, 条形码过程要求细胞是固定的, 因为它们需要温和地透条形码试剂 (钯同位素)24的插层。因此, 在本协议之后不能执行实时染色。应该注意的是, 活细胞条形码 (相对于这里描述的固定细胞条形码) 是一个可能性25,26, 但在这里的协议, 细胞是固定的, 以便他们可以存储和批处理一起。
批量抽样处理, 虽然从劳动和时间效率的角度来看, 也有自己的挑战。有了批量处理, 就需要精心策划的规范化过程。这种规范化过程可以在大规模细胞术工作流的不同步骤中解决。首先, 抗体对细胞数的浓度应保持一致的跨批次。我们发现, 有效的方法来达到这种一致的浓度是 1) 仔细滴定的抗体浓度的细胞数, 和 2) 标准化的每个样本的细胞数, 条形码在一起 (即, 100万每个样本的细胞, 所有20条形码样本)。其次, 为了解释仪器信号强度变异性 (不同的调整时间, 校准, 和信号衰减在运行时), 样品应悬浮和分析的质量细胞仪与适当的规范化珠解决方案 (表的材料), 其次是文件规范化后, 数据采集27 (但在 debarcoding 之前)。在整个条形码文件被规范化使用 Zunder 和为何芬克上校中描述的工具后, 单个示例 debarcoded. 24第三, 为了说明人工抗体染色技术的变异性, 我们建议将一个 "锚" 样本包含在每一个研究的条形码集上,即一个来自一个捐赠者 (人类研究) 的单一样本和与其他研究样本的处理方式相同。该样本应生成一次足够大的数量, 以分发到每个条码集分析的研究。这些从每个条码的锚样样本将被用来产生一个方差系数, 以纠正在整个研究样本的信号变异性的条形码 (即, 从条形码8的锚样本将用于纠正在条形码8的可变性)。
另一个重要的优化步骤是大规模细胞术抗体组的组装, 它将评估相关的免疫细胞亚群和细胞因子。抗体面板组件中的一些关键变量包括克隆的选择 (上面提到的)、最小背景信号的抗体效价、抗体靶点的选择和基于抗原丰度和通道灵敏度的同位素通道。补偿 "基于同位素杂质和氧化的信号溢出, 以及个体抗体的大量变异性。这些优化步骤在别处28 , 而不是在此处描述的协议范围内处理。
利用这种单细胞蛋白质组学方法研究外周血样本免疫异常, 可以确定免疫细胞亚群的异常表型, 无论是群体频率差异还是表达的变化特定的细胞表面标记 (如细胞活化的标记)。此外, 还可以识别免疫细胞子集内的功能异常, 如细胞因子的下或生产过剩。最后, 在没有外源性药物 (T6) 的6小时孵化后, 将样品立即固定 (T0) 与固定的过程进行比较, 说明了导致免疫细胞类型和细胞因子异常 (T6-T0) 的 "内在" 致病过程。这种方法可以针对各种系统性免疫介导的过程进行研究, 并根据刺激剂参与多种免疫细胞子集和通路。

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 &#956;M</td>
		</tr>
		<tr>
			<td>R848</td>
			<td>Invivogen</td>
			<td>tlrl-r848-5</td>
			<td>Stock Conc: 1 &#956;g/&#956;L; Final Conc: 1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>LPS-EK</td>
			<td>Invivogen</td>
			<td>tlrl-eklps</td>
			<td>Stock Conc: 1 &#956;g/&#956;L; Final Conc: 0.1 &#956;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>&#160;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: &#62;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 &#956;M; Final Conc: 0.1 &#956;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 TM 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&#937;.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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies used for Mass Cytometry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surface markers</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>Cytokines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1&#945;</td>
			<td>Biolegend</td>
			<td>364-3B3-14</td>
			<td>Mass: 147</td>
		</tr>
		<tr>
			<td>IL-1&#946;</td>
			<td>Biolegend&#160;</td>
			<td>H1b-98</td>
			<td>Mass: 169</td>
		</tr>
		<tr>
			<td>IL-1RA</td>
			<td>Santa Cruz&#160;</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&#160;</td>
			<td>C8.6</td>
			<td>Mass: 171</td>
		</tr>
		<tr>
			<td>IL-17A</td>
			<td>Biolegend&#160;</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&#946;</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&#945;</td>
			<td>Miltenyi&#160;</td>
			<td>LT27:295</td>
			<td>Mass: 175</td>
		</tr>
		<tr>
			<td>IFN&#947;</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&#945;</td>
			<td>Biolegend</td>
			<td>Mab11</td>
			<td>Mass: 166</td>
		</tr>
		<tr>
			<td>Note: 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&#160; Kit (Fluidigm Cat: 201300) according to manufacturer protocol.&#160;</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) 和内质 (R848, TLR7/8 激动剂) 激动剂。其他刺激剂包括佛波 12-酯 13-醋酸酯和 Ionomycin (PMAIONO), 可作为 T 细胞活化剂。特异的细胞因子也可以作为刺激剂, 以及其他特定的 B 和 T 细胞抗原。
为了演示本协议中描述的实验方法,图 2、图 3和图 4显示了使用 LPS、R848 和 PMAIONO 作为刺激条件获得的代表性结果。虽然协议中没有描述 PMAIONO 的使用情况, 但图 3和图 4显示了不同 (和选择性) 细胞类型和细胞因子在反应 TLR 激动剂 (LPS 和 R848) 与 T细胞激活剂 (PMAIONO)。考虑到26表面标记 (材料表) 被用来标定多个先天和自适应免疫细胞子集, 不同的 T、B、NK、单核细胞和树突状体细胞子集可以在单个样本中同时进行研究。此外, 还包括免疫细胞活化的标志物, 如 CD86 或 ICOS B 细胞和 PD1 T 细胞。图 2显示了一个具有代表性的有限门策略, 用以证明 CD14hi 单核细胞的评估。然而, 进一步详细和扩大的 T, B, NK, 单核, 树突, 和粒细胞子集的门, 可以发现在我们以前发表的工作在 O ' 戈尔曼和谢等等和 o ' 戈尔曼和香港等。14,15此外, 未经监督的分析方法包括 (不限于) 锹16、visNE17、柑橘18、Phenograph19和 Xshift20可用于评估海量细胞术数据 (不这里讨论)。以 CD14hi 单核细胞和 CD4 T 细胞为代表的先天和自适应免疫细胞亚群, 在这些细胞类型中显示细胞因子诱导的数据如图 3所示。选择数量的细胞因子, 以表明 R848 有选择地诱导 IL-12 (p40 亚基) 和 MCP1 在 CD14hi 单核, 而 LPS 没有 (图 3)。PMAIONO, 一个强有力的 T 细胞激活剂不诱导细胞因子在 CD14hi 单核细胞 (图 3), 但确实诱发干扰素伽玛 (IFNγ) 和肿瘤坏死因子α (TNFα) 在 CD4 T 细胞 (图 4)。成功的外周血样品刺激和处理技术将展示细胞因子诱导的模式, 特定于刺激条件和免疫子集, 如图 3和图 4所示。对于在26表面标记中捕捉到的细胞类型中所描述的14种细胞因子的完整分析, 请见 o ' 戈尔曼和谢等, 以及 o ' 戈尔曼和香港等。14,15
为了演示本议定书中描述的实验方法如何捕捉到系统性免疫性疾病 (如 SLE) 与健康捐献者相比的 "内在" 致病状态,图 5说明了细胞因子诱导 (Mip1β和MCP1) 在 CD14hi 单核细胞中发现的病外周血样本仅 (B 部分), 在没有任何外源刺激 (T6 相比 T0)。这些细胞因子通过 JAK 抑制治疗 ruxolitinib (T6 + 5R 比 T6) 在患病的外周血样本中 "调制/减少" (B 部分)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57780/57780fig1.jpg"> 图1。大鼠外周血刺激和处理的实验工作流程.红细胞裂解和固定的疾病外周血样本立即收集 (T0), 或后6小时孵化37°c 与蛋白质转运抑制剂 (公共交通) 在没有任何外源性药物 (T6), 或与 lps (T6 + lps), R848 (T6 + R848;蛋白转运抑制剂孵化仅为4小时), 或 ruxolitinib (T6 + 5R)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57780/57780fig1large.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/57780/57780fig2.jpg"> 图2。CD14hi 单核细胞识别的典型门控策略.在固定和红细胞裂解后, 来自健康捐献者的单个裂解/固定细胞样本条形码, 标记有26抗体, 对表面标记, 透和染色14抗体抗细胞因子 (材料表)。给出了代表 CD14hi 单核细胞识别的有限门策略。从左到右, 所表示的每个2D 图形都是从左侧的2D 图形中被门控 (蓝色框) 的父填充的填充子集。在 o ' 戈尔曼和谢等, 和 o ' 戈尔曼和香港等, 可以找到一个扩展的门控策略。14,15<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57780/57780fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57780/57780fig3.jpg"> 图3。CD14hi 单核细胞中细胞因子诱导的例子在 LPS、R848 和 PMAIONO 的刺激下.以静态 (T0)、R848 (T6+R848) 和 PMAIONO (T6+PMAIONO) 为例, 对健康献血者的外周血标本进行了典型的大规模细胞仪分析。CD14hi 单核细胞中细胞因子诱导的一个例子;所选择的细胞因子与刺激剂的特异性 (TLR 激动素与 T 细胞活化剂) 被描述。扩展数据为所有细胞因子诱导跨多个免疫细胞亚群确定与这个质量细胞术抗体小组可以发现在 o ' 戈尔曼和谢等, 和 o ' 戈尔曼和香港等。14,15<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57780/57780fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57780/57780fig4.jpg"> 图4。CD4 T 细胞细胞因子诱导的例子在 LPS、R848 和 PMAIONO 刺激之后.以静态 (T0)、R848 (T6+R848) 和 PMAIONO (T6+PMAIONO) 为例, 对健康献血者的外周血标本进行了典型的大规模细胞仪分析。CD4 T 细胞细胞因子诱导的例子显示;所选择的细胞因子与刺激剂的特异性 (TLR 激动素与 T 细胞活化剂) 被描述。扩展数据为所有细胞因子诱导跨多个免疫细胞亚群确定与这个质量细胞术抗体小组可以发现在 o ' 戈尔曼和谢等, 和 o ' 戈尔曼和香港等。14,15<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57780/57780fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57780/57780fig5.jpg"> 图5。系统性红斑狼疮患者 CD14hi 单核细胞因子诱导的例子。对健康捐献者 (A) 和 SLE 患者 (B) 的外周血标本进行了典型的大容量细胞仪分析。CD14hi 单核细胞因子诱导的例子;选定的细胞因子与系统性红斑狼疮疾病 "内在" 致病过程 (T6) (即诱导 MIP1β和 MCP1 仅在 sle 患者), 和免疫调节剂 ruxolitinib (T6 + 5R) 的影响描述。扩展数据为所有细胞因子诱导跨多个免疫细胞亚群确定与这个质量细胞术抗体小组可以发现在 O ' 戈尔曼和香港等。15<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57780/57780fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.3K 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

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

科研

免疫与感染

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

鞘糖脂抗原的质谱分析

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

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 ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

细胞因子产生的病毒逃避解剖先天免疫信号

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

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 ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

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 ...

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 ...