所有显微术都是在萨克雷的凯克显微镜中心进行的, 并得到了 S10OD016240 的支持。S.L.F. 得到 NIH K08AI119142 和 R21AI130281 的支持。我们感谢理查德 Flavell 博士和布拉德库克森博士为 caspase-1/11 不足的老鼠, 和布拉德博士库克森共享实验室设施和设备。

所有动物程序均经华盛顿大学动物保育和使用委员会指南批准并进行。
1. 骨髓的收获
<ol><li>灌装从老鼠身上取出股骨和胫骨, 用无菌器械清洁骨骼。将被清洗的骨骼放在 Dulbecco 修饰的鹰培养基 (DMEM) + 5 毫米 HEPES + 0.2 毫克/毫升 l-谷氨酰胺 + 0.05 毫米 2-巯基乙醇 + 50 毫克/毫升庆大霉素硫酸盐 + 1万毫升青霉素/链霉素 + 10% 胎牛血清 (血清, DMEM-10 完成) 和孵化在冰上管15分钟。</li>
	<li>使用剪刀去除近端和远端球关节。插入一个25克针连接到一个10毫升注射器填充中的骨骼空心。用 DMEM-10 完成骨髓冲洗。并用重悬任何团块通过吹打中上下轻轻地。</li>
	<li>在 300 x g 上离心细胞10分钟, 用 hemocytometer 计数细胞数。在这一点上, 要么冻结的细胞使用90% 的血清 + 10% 二甲基亚砜 (亚砜) 或板块的细胞在15厘米培养皿, 以区分巨细胞使用 L929 条件培养基 (步骤 2.1)。</li>
</ol>2. 骨髓源性巨噬细胞的分化
<ol><li>使用预热分化培养基 (21 毫升的 DMEM-10 完整和9毫升的 L929 细胞条件培养基, 如... 18), 将骨髓细胞放入15厘米非组织培养处理的培养皿 (1.2-1.5 x 107细胞)。在37摄氏度和 5% CO2上孵化出7天的板材。在3或4天增加30毫升的分化培养基到盘子里。 注: 重要的是不要使用组织培养治疗板, 因为巨噬细胞将难以分离和收获。</li>
	<li>收集巨噬细胞, 用磷酸盐缓冲盐水 (pbs) 冲洗盘子, 并加入20毫升的冷 PBS + 1 毫米乙二胺四乙酸 (EDTA)。在4摄氏度孵化出10分钟的盘子, 通过将 pbs + EDTA 吹打在细胞上, 用10毫升的 pbs 冲洗盘子。将两个洗涤液结合成两个15毫升锥形管。</li>
	<li>离心细胞在 300 x g 10 分钟和并用重悬10毫升的苯酚-红色自由 DMEM + 5 毫米 HEPES + 0.2 毫克/毫升 l-谷氨酰胺 + 0.05 毫米 2-巯基乙醇 + 5% 血清 (DMEM-5) 和计数的细胞使用 hemocytometer 或库尔特计数器。在计数期间把细胞放在冰上。</li>
	<li>将组织培养的巨噬细胞种子涂在 2 x 105细胞/毫升上。对于显微实验, 种子1毫升的细胞在盖玻片24井板。乳酸脱氢酶释放化验, 种子100µL 的细胞在96井板。对于乳酸脱氢酶测定, 种子9井 (3 井未处理, 3 井与100% 溶解控制和3个井为实验性刺激)。</li>
	<li>离心96井板5分钟在 300 x g, 使确定的细胞均匀分布在井。</li>
	<li>在启动启动过程前, 在37°c + 5% CO2上隔夜孵化盘。</li>
</ol>3. 启动
<ol><li>用新鲜介质 (DMEM-5) 取代含有 100 ng/毫升 LPS (500 ul 为24井板或 50 ul 的96井板) 的介质。 注: 脂多糖有多种结构变化, 影响刺激 TLR4-mediated 启动的能力19。来自明尼苏达州沙门氏菌R595 (Re) 的 LPS 可从多个供应商处获得, 建议使用。</li>
	<li>在37°c 和 5% CO2上孵化3小时的板材。</li>
</ol>4. 激活 NLRP3 Inflammasome 与尼日利亚菌素和标记与 YVAD (芬兰马克
<ol><li>取出培养基, 用 290 ul DMEM-5, 其中含有5微米尼日利亚菌素和5毫米甘氨酸。添加甘氨酸可减少在 caspase-1 激活5期间发生的细胞裂解量。孵育60分钟在37°c 和 5% CO2。使用野生类型和 caspase-1 缺乏巨噬细胞, 以确保所观察到的标记是特定的 caspase-1。</li>
	<li>在最后45分钟, 添加 10 ul 30x YVAD-(芬兰马克, 根据制造商的指示编写。</li>
	<li>60分钟后, 取出培养基, 用1毫升的冷 PBS 清洗细胞三次5分钟。</li>
	<li>添加 250 ul 2% 多聚甲醛每井和孵化在冰覆盖铝箔为30分钟。</li>
	<li>在最后5分钟, 添加 4 ul 0.2 毫米远红色荧光核酸染色标签的细胞核。4 ', 6-diamidino-2-苯基吲哚 (DAPI) 或其他染料也可以用来标记 DNA。</li>
	<li>用1毫升的冷 PBS 冲洗细胞三次, 用 7 ul 防褪色安装介质将盖玻片安装到显微镜幻灯片上。让安装介质硬化过夜之前, 用指甲油密封盖玻片到显微镜幻灯片。</li>
	<li>用共焦显微镜对巨噬细胞进行成像。YVAD-(芬兰马克的前/Em 是492/520 毫微米和642/661 的远红色荧光核酸染色。一定要建立显微镜, 使未经处理的细胞不显示任何背景染色的488通道。否则, 将检测到背景 YVAD-(芬兰马克染色, 而不是实际的活动 caspase-1。 注: 显微镜设置在6节中描述。</li>
</ol>5. 抗体染色
注意: 用抗体对细胞进行标记, 以检测 ASC、种子、素数, 并使细胞与上一节尼日利亚菌素相同。随后的处理如下:
<ol><li>用1毫升的冷 PBS 清洗细胞三次, 每5分钟。添加 250 ul 的固定和通透溶液。在冰上孵化细胞, 覆盖30分钟。</li>
	<li>用1毫升的洗涤缓冲器将巨噬细胞清洗三次, 每次5分钟。</li>
	<li>添加 250 ul 的主要抗体对 ASC 稀释1:500 在洗涤缓冲和孵化1小时冰。包括一个不接收任何主要抗体的盖玻片, 但只接受辅助。此盖玻片将在显微镜安装过程中使用, 以设置正确的偏移量。</li>
	<li>用1毫升洗涤缓冲器, 每三次清洗细胞5分钟。</li>
	<li>添加 250 ul 二次抗体 (荧光染料共轭山羊抗鼠) 稀释1:500 在洗涤缓冲和孵化 1 h 在冰上。添加 4 ul 0.2 毫米远红色荧光核酸染色的最后5分钟的孵化, 以标记细胞核。</li>
	<li>使用 7 ul 防褪色安装介质, 用1毫升的冷水洗涤缓冲液将细胞盖玻片, 并将其装入显微镜幻灯片上。让安装介质硬化过夜之前, 密封盖玻片到显微镜幻灯片使用明确的指甲油。</li>
	<li>图像巨噬细胞的共聚焦显微镜。继发抗体的前/Em 为 555/580 nm 和642/661 的远红色荧光核酸染色 (如下所述)。 注: 用 YVAD-(芬兰马克和抗体标记进行标记可以组合显示主动 caspase-1 和 ASC 的联合定位。为此, 请遵循 YVAD (芬兰马克标记的条件, 然后切换到抗体标记协议进行进一步处理。</li>
</ol>6. 用共焦显微镜成像标记巨噬细胞
注: 染色细胞可以在盖玻片被允许治疗一夜之间, 并已被密封到显微镜幻灯片与指甲油。这里描述了用共焦显微镜成像。细胞也可以通过标准荧光显微镜查看。
<ol><li>将幻灯片与未经处理的控制单元放在显微镜阶段, 并将显微镜聚焦在细胞上。</li>
	<li>使用显微镜查找表 (查找) 设置, 调整偏移量, 使未处理的细胞中没有阳性的 YVAD (芬兰马克染色。这一过程也可以使用 caspase-1 缺乏的巨噬细胞进行。 注意: 对于实验中使用的每个通道, 偏移量必须进行调整, 但是设置正确的偏移量对于 YVAD-(芬兰马克通道和荧光抗体通道是最关键的。DNA 通道的偏移量不太重要。设置偏移量后, 不要为实验的其余部分调整此设置。</li>
	<li>将幻灯片切换到尼日利亚菌素处理的示例。找到一个包含两个阳性和阴性的细胞 YVAD-(芬兰马克染色的字段。将 YVAD-(芬兰马克通道的成像平面调整为具有最大阳性染色强度的平面。这通常是 inflammasome 焦点的中心被成像的平面。</li>
	<li>调整增益, 使病灶在有凝聚细胞核的细胞中可见。设置增益和偏移量后, 将这些设置保留在成像会话的其余部分。 注意: 一个简单的方法来确定一个细胞是否是 pyroptotic 是通过观察核形态。有活性 caspase-1 的细胞有圆形和凝聚的细胞核, 而核仁在细胞中可见, 而不 caspase-1 活化, 细胞核形态与未经处理的细胞相似。</li>
	<li>以100X 的总放大倍数收集5个随机选定字段的图像。这通常会导致每个图像大约40个单元格。对每个示例重复此过程。</li>
	<li>使用图像分析软件计算每个图像中的单元格数。然后计算有阳性 YVAD (芬兰马克染色的细胞数。表示 caspase-1 激活级别为 YVAD (芬兰马克阳性细胞的百分比。</li>
</ol>7. 乳酸脱氢酶释放试验
注: 对于 LDH 释放试验, 种子和素数的细胞如前一节所述, 除了使用96井板。启动后请勿取出介质。
<ol><li>添加 50 ul 的 DMEM-5 包含10微米尼日利亚菌素到实验井和 50 ul DMEM-5 到自发和100% 裂解控制井。不要添加甘氨酸在这个实验, 因为目标是测量细胞裂解。孵育60分钟在37°c 和 5% CO2。</li>
	<li>30分钟后, 将 10 ul 10x 裂解缓冲液添加到100% 裂解控制井中, 并将 10 ul 的 DMEM-5 添加到其他井中。在这种孵化过程中, 除去1小瓶的基质混合物 (1.5 毫升的冷冻干燥基质) 和1整除的化验缓冲器 (~ 12 毫升) 从冰箱, 让他们解冻保护, 从光到室温。 注: 衬底混合物含有四氮唑盐 (iodonitro-四氮唑紫罗兰), 它被转换为红色 formazan 产品。</li>
	<li>在60分钟的总孵化之后, 离心板5分钟在 500 x g 在10°c。将上清的 50 ul 转移到一个清晰的平底板上。在此期间, 在使用前, 将12毫升的化验缓冲器添加到基质混合物的瓶子中, 在室温下孵育5分钟。</li>
	<li>在每个井上加 50 ul 的基底, 在黑暗中孵化30分钟。仅包含空白值的中等井。检查15分钟后的车牌, 确保信号不会超过车牌读取器的检测限制。</li>
	<li>30分钟后, 添加 50 ul 的停止溶液 (1 米乙酸) 和测量 OD490使用板读取器。</li>
	<li>使用以下公式计算细胞裂解的百分比: % Cytoxicity = (实验性自发)/(最大自发)) ×100 实验: 尼日利亚菌素治疗细胞;自发的: 未经治疗的细胞;最大值: 暴露在溶解缓冲中的细胞</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+and+cell+lysis+are+separable+events+during+pyroptosis.">Cell death and cell lysis are separable events during pyroptosis.</a> Cell Death Discov. , (2017).</li><li>Russo, H. 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=Active+Caspase-1+Induces+Plasma+Membrane+Pores+That+Precede+Pyroptotic+Lysis+and+Are+Blocked+by+Lanthanides.">Active Caspase-1 Induces Plasma Membrane Pores That Precede Pyroptotic Lysis and Are Blocked by Lanthanides.</a> J Immunol. 197 (4), 1353-1367 (2016).</li><li>Evavold, C. L., 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+Pore-Forming+Protein+Gasdermin+D+Regulates+Interleukin-1+Secretion+from+Living+Macrophages.">The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages.</a> Immunity. , (2017).</li></ol>

作者没有利益冲突披露

在这篇手稿中, 我们提出了两种技术来检查 inflammasome 活化和 NLRP3 刺激后 caspase-1 活化的结果在小鼠骨髓源性巨噬细胞。第一种技术允许研究者使用荧光报告器 YVAD-(芬兰马克来确定细胞基础上的 caspase-1 活化水平。这个记者是高度特异的结合 caspase-1 家族的酶, 看不见染色 caspase-1/11 缺乏的巨噬细胞。特异性也显示了 LaRock et al。在他们的手稿中描述了耶尔森氏菌蛋白 YopM 对 caspase-1 活化...

Inflammasome 介导的炎症是防御病原生物的一个关键组成部分1, 但也构成许多疾病的病因2。炎症反应的广泛感染是由胞浆检测病原体相关的分子模式 (PAMPs) 或损害相关的分子模式 (抑) 触发。模式识别受体 (PRR), 包括点头样受体 (NLR) 家族的成员, oligomerize 在这些 PAMPs 和抑的检测。这触发了一个多蛋白复合体的形成, 称为 inflammasome, 其中包含 PRR, 适配器蛋白凋亡相关的斑点样蛋白, 含有 C 终端 caspase-招聘领域 (ASC), 和赞成形式的caspase-13,4。这一复杂的 caspase-1 允许接近诱导的自动激活。活跃的 caspase-1 然后导致一组事件是炎症细胞死亡途径 pyroptosis 的特征。这些事件包括: 炎症细胞因子 IL-1β和 IL-18 的分裂和释放, 溶酶体胞吐作用释放溶酶物的内容进入细胞外空间, 核冷凝和 gasdermin D 分裂。释放的 N 端域 gasdermin D 插入到等离子膜, 形成毛孔, 导致等离子膜破裂和释放炎症的内容, 除了带走一个保护生态位的病原体复制5,6,7。
在这里, 我们专注于研究良好的 NLRP3 inflammasome。NLRP3 inflammasome 的激活是通过两步过程8进行的。第一个 "启动" 步骤是通过对一种微生物产品的类似收费受体 (TLR) 的识别而产生的。这是复制在实验室设置使用 LPS 刺激 TLR4。这种刺激上调 NLRP3 和 pro-IL-1β通过 NF-kB 信号。另外启动许可证NLRP3 通过非转录机制诱导其 deubiquitination9,10和磷酸化或除磷的特定残留 11, 12。
NLRP3 激活的第二个信号被认为涉及线粒体因素、活性氧种类、钾外流和钙信号, 尽管 NLRP3 激活的统一机制仍然难以捉摸8。激活 NLRP3 oligomerizes 通过其晚上域之间的交互, 并通过 pyrin (PYD) 域13的绑定来招募 ASC。在每个细胞中, 这些大分子复合物形成一个单显微镜可见的焦点。ASC 最初被确定为 22 KDa 蛋白, 在人类白血病细胞凋亡期间形成 "斑点", 并命名为凋亡相关的斑点状蛋白, 含有卡14。后来确定, asc 新兵 pro-caspase-1 通过与 pro-caspase-1 卡的互动, 形成 inflammasome15。
并非所有 NLRs 都需要 ASC 的存在来诱导 caspase-1 激活。与 NLRP3 不同, NLRC4 和 NLRP1b 具有卡域, 可以通过卡卡交互直接招聘 pro-caspase-1, 以诱导 caspase-1 激活。在没有 ASC 的情况下, 活跃的 caspase-1 在整个细胞质中仍然弥漫, 并不形成一个单一的焦点。这种弥散活性 caspase-1 足以诱发 pyroptotic 细胞死亡, 但无法处理 pro-IL-1β13,16。
在这篇手稿中, 我们将讨论两种评估 inflammasome 激活的方法。第一个使用荧光活性探针, YVAD-(芬兰马克, 它结合了 caspase-1 家族的蛋白酶。这个家庭包括 caspase-1, 还有老鼠 caspase-11 和人类 caspase-4 和 caspase-5。通过在所有实验中使用 caspase-1/11 缺陷小鼠的巨噬细胞, 可以解决该探针的特异性。该方法可与 inflammasome 成分的抗体标记相结合, 并对其进行描述。显微可视化可以识别含有活性 caspase-1 和 oligomerized 的单个细胞。利用这一方法, 研究人员将能够确定在 inflammasome 形成级联他们的宿主细胞的操作或所研究的致病有机体的作用。例如, 可以区分某一特定的干预是否阻止了 NLRP3 复合体的招募, 或者是针对随后的 caspase-117的招募和激活。检查 caspase-1 激活的结果, 如 IL-1β分泌物, 将无法区分这两种可能性。此外, IL-1β的分泌可以改变, 而不改变细胞激活 caspase-1 的能力, 并接受 pyroptotic 细胞死亡16。
第二种方法测量乳酸脱氢酶 (LDH) 的释放到细胞上清, 这是发生在 pyroptotic 巨噬细胞裂解后, caspase-1 激活, 如上文所述。这第二种方法是以人口为基础的方法, 因为在整个油井中乳酸脱氢酶的释放量被测量。这种简单的方法允许快速分析 (30 分钟的潜伏期) 的样本, 以确定是否有 caspase-1-mediated 细胞死亡, 并可以执行96井板格式。
这些方法是互补的, 每个都有不同的优点, 而且两者都易于修改。例如, 在 inflammasome 刺激之前, 用靶向小分子量化合物的巨噬细胞治疗可以用来研究某些蛋白质在控制炎症反应方面可能具有的作用。

<table>
	<tbody>
		<tr>
			<td>E-MEM</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td>For growing L929 cells</td>
		</tr>
		<tr>
			<td>NCRC clone 929 (L929)&#160;</td>
			<td>ATCC</td>
			<td>CCL-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation/Permeabilization Kit</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Includes fixation and permeabilization solution, and wash buffer.&#160; Proprietary formulations. Contains 4.2% formaldehyde</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Biorad</td>
			<td>161-0718</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytotox96 Nonradioactive cytotoxicity assay</td>
			<td>Fisher Scientific</td>
			<td>PR-G1780</td>
			<td>Includes Substrate Mix and Assay Buffer; Proprietary formulations. Stop solution is 1 M Acetic acid</td>
		</tr>
		<tr>
			<td>FAM-YVAD-FMK</td>
			<td>Immunocytochemistry Technologies</td>
			<td>98</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, no glutamine</td>
			<td>Invitrogen</td>
			<td>11960044</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, no glutamine, no phenol red</td>
			<td>Invitrogen</td>
			<td>31053028</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS, no calcium, no magnesium</td>
			<td>Invitrogen</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, US origin</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td>Heat inactivated at 55&#176;C for 50 min</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Invitrogen</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Mounting Medium</td>
			<td>Invitrogen</td>
			<td>p36934</td>
			<td>Proprietary formulation. Curing mounting medium. Hardens to refractory index of 1.46</td>
		</tr>
		<tr>
			<td>goat anti-mouse ALEXA555</td>
			<td>Invitrogen</td>
			<td>A-21422</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (Ultra Pure)</td>
			<td>Invitrogen</td>
			<td>11344041</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>21051024</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>TO-PRO-3 Iodide</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>far-red fluorescent nucleic acid stain&#160;</td>
		</tr>
		<tr>
			<td>C57BL/6J mouse</td>
			<td>Jackson Laboratoy</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>caspase-1/11-/- mouse</td>
			<td>Jackson Laboratory</td>
			<td>016621</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8X Confocal Microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure LPS from Salmonella minnesota R595 (Re)</td>
			<td>List Biologicals</td>
			<td>434</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ASC clone 2EI-7</td>
			<td>Millipore-Sigma</td>
			<td>04-417</td>
			<td></td>
		</tr>
		<tr>
			<td>beta-mercapto-ethanol</td>
			<td>Millipore-Sigma</td>
			<td>M6250-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Millipore-Sigma</td>
			<td>D2650-5X10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Millipore-Sigma</td>
			<td>E5391</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectramax M3 plate reader</td>
			<td>Molecular Devices</td>
			<td>5000414</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of inflammasome activation and pyroptotic cell death in murine bone marrow-derived macrophages

Inflammasomes 是先天免疫信号平台, 是成功控制许多致病有机体所必需的, 同时也促进炎症和 autoinflammatory 疾病。Inflammasomes 由胞浆模式识别受体激活, 包括点头样受体 (NLR) 家族的成员。这些受体 oligomerize 对微生物或损伤相关刺激的检测。随后, 适配器蛋白 ASC 的招募形成了一个显微镜可见的 inflammasome 复合体, 通过接近诱导的自动激活激活 caspase-1。活化后, caspase-1 pro-IL-1β和 pro-IL-18, 导致这些促炎性细胞因子的活化和分泌。Caspase-1 还介导细胞死亡的炎症形式称为 pyroptosis, 其特征是膜完整性的丧失和细胞裂解。Caspase-1 gasdermin D, 释放形成等离子膜孔隙的 N 末端片段, 导致渗透裂解。
体外, caspase-1 的活化可以通过标记骨髓源性巨噬细胞与 caspase-1 活动探针 YVAD (芬兰马克来确定, 并通过对适配器蛋白 ASC 的抗体对细胞进行标记。这种技术可以通过荧光显微镜识别单个细胞中的 inflammasome 形成和 caspase-1 活化。Pyroptotic 细胞死亡可以通过测量胞浆乳酸脱氢酶释放到培养基中来检测。该程序简单, 成本效益高, 并以96井板格式进行, 允许适应筛选。在这篇手稿中, 我们展示了 NLRP3 inflammasome 的活化尼日利亚菌素导致了适配器蛋白 ASC 和主动 caspase-1 的共同定位, 导致 pyroptosis。

为了检测暴露于尼日利亚菌素后的 caspase-1 激活, 细胞被处理如协议部分所述。我们结合了 YVAD-(芬兰马克和 ASC 抗体标记, 以表明 ASC 和主动 caspase-1 合作本地化 NLRP3-mediated inflammasome 激活。图 1A显示, 暴露于5微米尼日利亚菌素的野生型巨噬细胞在核周地区形成了 ASC 聚焦。这些病灶也包含活跃的 caspase-1 的共同本地化的 ASC 与 YVAD-(芬兰马克。积极 caspase-...

Watch this Scientific Journal Video about Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages at JoVE.com

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

我们描述了 NLRP3 inflammasome 激活的细胞基础上的荧光显微镜和染色的主动 caspase-1 和适配器, ASC。以乳酸脱氢酶释放试验为依据, 检测 pyroptotic 裂解的人群。这些技术可以适应研究 inflammasome 生物学的许多方面。

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

detection-inflammasome-activation-pyroptotic-cell-death-murine-bone

Research

JoVE Journal

Immunology and Infection

10.5K 次观看.  University of Washington. 我们描述了 NLRP3 inflammasome 激活的细胞基础上的荧光显微镜和染色的主动 caspase-1 和适配器, ASC。以乳酸脱氢酶释放试验为依据, 检测 pyroptotic 裂解的人群。这些技术可以适应研究 inflammasome 生物学的许多方面。

视频: Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

科研

免疫与感染

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

激活和NLRP3炎性活性的人单核细胞来源的树突状细胞用IL-1β测量

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

抗体药物对小鼠骨髓及腹腔巨噬细胞活化作用的评价

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...