1. 克隆和细胞线生成
<ol><li>PCR 放大 NBB 区域, 对应于氨基酸残滓 1-140 (NBB140), 从人的 MLKL cDNA 的框架标准限制酶的克隆与齐聚的领域 2x FK506 结合蛋白 (2xFKBP 或 2xFV) 和金星荧光蛋白入强力霉素 (Dox) 诱导的逆转录病毒载体 pRetroX-TRE3G 获得 NBB140-2 xfv-金星 (表 1, 图 1 a B)。</li>
	<li>用 MEFs 抗原瞬态转染法从绿色实验室中获得的不朽初级mlkl-/或ripk3- mlkl/ 小鼠胚成纤维细胞 (SV40-large)表达质粒。在 DMEM 周内选择永生化细胞, 并在标准组织中以10% 胎牛血清 (血清)、2毫米 l-谷氨酰胺、100的 U/毫升青霉素和链霉素、1毫米丙酮酸钠和不必要氨基酸在37°c 和 5% CO2中补充。文化孵化器。</li>
	<li>传感器 SV40-immortalized mlkl- MEFs 与反式四环素控制 transactivator (rtTA) 包含的逆转录酶表达的质粒含有 blasticidin 抗性基因 (我们的改良质粒) 和治疗1周与5微克/毫升 blasticidin 选择的细胞稳定表达 rtTA28。</li>
	<li>传感器 rtTA 表达 MEFs 与 Dox 诱导的逆转录病毒载体含有嵌合体 MLKL (pRetroX NBB140-2 xfv-金星普洛), 并选择使用4微克/毫升嘌呤霉素一周, 2 天后转导。</li>
</ol>2. MLKL 介导 Necroptosis 的活体细胞显微成像
<ol><li>板mlkl- MEFs 表示 NBB140-2 xfv-金星密度5万细胞每井 (1 毫升/井) 在24井板, 和孵化过夜 (图 2a)。使用活细胞染色自动计数 (见材料表)。</li>
	<li>用 Dox (0.5 微克/毫升) 治疗细胞12小时, 以诱导 NBB140-2 xfv 金星的表达。</li>
	<li>诱导 necroptosis 与25毫微米 FKBP dimerizer (暗淡) 除25毫微米膜不透水绿色荧光染料 (参见材料表) 在标准 DMEM (参见 1.2)。细胞 necroptosis 积累染料, 因为他们的等离子膜完整性被 NBB140介导的破裂破坏。用三份或四份进行化验。</li>
	<li>使用单或双色成像系统监视 necroptosis (见材料表), 将血管放置在哺乳动物组织培养孵化器中的相应成像单元中。</li>
	<li>打开软件 (参见材料表), 然后在 "任务" 列表下选择选项卡计划即将进行的扫描。 注意:此协议用于使用单色系统进行成像, 但双色系统可使用类似的软件。</li>
	<li>在 "属性" 选项卡中, 选择 "物理布局" 选项卡和 "快速荧光" 中的 "托盘、容器、扫描类型" 和 "扫描模式"。</li>
	<li>通过单击 "时间线" 窗口和获取时间点和频率的总次数 (通常为 6 h 到 12 h, 或每5分钟为快速动力学 necroptosis) 添加 "扫描时间", 并通过选择 "应用程序" 来开始图像获取。"(红色按钮)。</li>
	<li>使用图像分析软件 (参见材料表), 通过从 "任务窗格" (necroptosis) 中选择 "对象计数新的分析, 将固定的分割阈值放在背景水平之上" 来量化, 这可以通过悬停鼠标光标悬停在背景区域的多个图像中使用的分析。</li>
	<li>通过选择使用当前图像预览并根据需要细化阈值级别来检查荧光对象。</li>
	<li>通过打开 "启动新分析" 选项卡, 选择 "时间范围", 选择要分析的井, 输入 "分析作业名称", 然后按 "确定", 将绿色荧光计数集成。</li>
	<li>通过双击相应的作业名称并从图形/导出窗口导出外部绘图软件中的其他分析, Access 从 "分析作业" 选项卡分析数据 (见材料表)。</li>
	<li>定量数据作为绿色荧光正 (+ ve) 计数/毫米2或正常化计数通过汇合并且表达数据作为绿色荧光 + ve 计数或汇合。</li>
	<li>有选择地, 在实验终点, 染色细胞与 100 nM 的膜-permeant 绿色荧光染料 (见材料表)。将数据规范化为% necroptotic 单元格, 作为端点上绿色荧光/绿色荧光的比值。</li>
</ol>3. MLKL 对血浆膜招募和通透的活体细胞共聚焦显微成像
<ol><li>板mlkl- MEFs 表示 NBB140-2 xfv-金星在密度2万个细胞每井 (0.5 毫升/井) 在玻璃底部 4-井显微镜室预处理与50µg/毫升纤维连接蛋白在 PBS 在37°c 为30分钟和孵化为 ~ 12 h (图 2B)。</li>
	<li>用 Dox (0.5 微克/毫升) 治疗细胞12小时, 诱导 NBB140-2 xfv 金星的表达。</li>
	<li>通过孵化与新鲜培养基 (1 毫升/井) 含有 25 nM 暗淡诱导 necroptosis 诱导 NBB140-2 xfv 金星活化和转运到等离子膜。</li>
	<li>将腔室放在旋转圆盘激光扫描共聚焦显微镜上, 安装有环境控制的倒立架和 63 1.4 NA 目标, 并使用选择软件开始图像采集 (见材料表)。</li>
	<li>初始化软件, 选择 "焦点" 图标和 YFP 过滤器配置 (激发波长 515 nm)。</li>
	<li>找到视图和焦点平面的字段, 并使用 "确定焦点" 选项卡参与焦点维护系统。</li>
	<li>要开始获取, 请选择 "捕获" 选项卡, 然后依次分配过滤器配置、适当的 EMCCD 相机曝光时间和增强增益, 以及时间推移采集参数, 包括间隔和采集长度。</li>
	<li>通过使用 515 nm 激光 (影片 1) 的 EMCCD 照相机, 在 necroptosis 期间通过获取图像, 监测荧光 NBB140-2 xfv-金星蛋白的细胞再分布。 注: 漂白是荧光蛋白成像的关注事项。金星是漂白29的抗性荧光蛋白之一。如果使用更容易漂白的荧光蛋白, 图像每三十年代或更长时间, 以允许恢复在成像时间点之间的信号。</li>
	<li>为了监测 NBB140-2 xfv-金星在 necroptosis 期间的等离子膜协会, 每年十年代, 图像在3.3 由全内反射荧光 (TIRF) 显微镜制备, 使用一个装有自动 TIRF 滑块的显微镜和100X 1.4 NA 目标和 EMCCD 照相机 (影片 2)。按照步骤 3.5–3.7, 但根据显微镜室的样品和底部玻璃厚度调整 TIRF 焦点标签内的 TIRF 角。 注: 在 TIRF 分析中, 等离子膜标记 (如 LCK-RFP) 可作为血浆膜定位的控制手段。</li>
	<li>用高斯函数的负归一化二阶导数 (马尔算法) 进行图像处理以提高卷积的质量。</li>
</ol>4. 电子显微术
<ol><li>mlkl MEFs 表达 NBB140-2 xfv-金星密度500万细胞在等离子涂层150毫米细胞培养皿和孵化为 12 h (图 3)。</li>
	<li>用强力霉素 (0.5 微克/毫升) 治疗细胞, 以诱导1-140-2 xfv-金星的表达为 12 h。</li>
	<li>诱导 NBB140-2 xfv-金星活化和易位到血浆膜与 25 nM homodimerizer 5 分钟。这一次是建立在 necroptosis 的动力学中观察到的活细胞成像。</li>
	<li>用10毫升的2.5% 戊二醛、2% 多聚甲醛在0.1 米常使用缓冲液 pH 7.4 (常使用缓冲) 中取出介质并固定细胞, 在37摄氏度前预热。</li>
	<li>收集样品的刮和旋转样品10分钟在 500 x g。放弃上清。</li>
	<li>在0.1 米钠常使用缓冲器中, 用1.5% 钾盐在减少的2% 锇毒气中对样品进行1.5 小时的后缀。锇毒气是一种广泛应用于 TEM 中的染色剂, 可提供对比度。在 necroptosis 细胞扫描电镜前, 我们用 TEM 进行了初步分析。</li>
	<li>将样品在超纯水中冲洗5次, 每 500 x克5 分钟, 然后在自动处理器上用缓冲液 (见 4.6)、水和乙醇漂洗。放弃上清。</li>
	<li>对于扫描电镜, 染色前固定样品与1% 醋酸铀和铅天门冬氨酸重金属染色31。</li>
	<li>通过一系列的酒精和丙烯氧化物溶液来脱水样品。</li>
	<li>将样品嵌入硬树脂32以获得树脂块。</li>
	<li>切厚部分0.5 微米, 以确定正确的分析区域。</li>
	<li>用导电金属 (铱) 的超薄薄膜涂上样品。</li>
	<li>图像和分析样本 (图 3)。为量化, 用电子显微镜扫描5凯文的树脂块表面与暴露的细胞的低放大图像。</li>
	<li>通过扫描电镜或成像软件捕获的单个图像中的细胞可视化, 可以将多个视场连接到一个连续的图像30中。通过在选择软件中生成高分辨率蒙太奇 (见材料表), 大约有100个细胞可以用这种方式来评估 necroptosis 细胞的分数。 注: 扫描电镜评分坏死形态学比较正常细胞特征与前坏死或坏死表型的进展。这些特征包括膜的改变, 包括微绒毛的消失, 扁平化, 细胞破裂从 10 nm 到1µm 的大小, 或失去的器官结构的至少30–40% 的胞浆细胞区。</li>
</ol>5. MLKL 的脂质结合核磁共振 (NMR) 光谱学
<ol><li>用标准蛋白表达和纯化制备15N 标记的 NBB1-156 , 如前所述24。</li>
	<li>溶解500µg 12-二硬脂酸甘油酯锡磷脂酰 3-肌醇磷脂铵盐 (18:0 PI) 和 12-dioleoyl-锡磷脂酰-3-磷-(1 '-肌醇) 铵盐 (18:1 PI) 在500µL 冰冷 CHCl3每个为最后的浓度1毫克/毫升。</li>
	<li>油脂实验1毫克/毫升18:0 和 18:1 PI 在超声波水浴为1分钟在室温或至多5分钟在冰水混合物。</li>
	<li>
整除1毫克/毫升18:0 和 18:1 PI 成清洁玻璃瓶为个别核磁共振滴定系列 (图 4)。用玻璃气密注射器在有机溶剂中转移脂质。
	<ol>
<li>整除132µL 1 毫克/毫升 18:1 PI (摩尔质量 = 880.137 克/摩尔) 在 CHCl3为最后泥沙容量1200µL 在125µM 集中。 注: 为5毫米核磁共振管, 准备连续稀释量 > 1000 µL 和最后核磁共振样品容量 > 500 µL。为3毫米核磁共振管, 准备连续稀释容量 > 300 µL 和最后核磁共振样品容量 > 150 µL。</li>
	</ol>
</li>
	<li>整除1毫克/毫升猪脑 L-α-phosphatidylinositol-4,5-bisphosphate 铵盐 20:9: 1 CHCl3/甲醇/小时2O (脑 PI (45) P2) 成清洁玻璃瓶。</li>
	<li>将小瓶置于氩气气流 (Ar) 或氮气 (N2) 中, 适度流动, 蒸发有机溶剂, 不溅到玻璃玻璃瓶壁上。五到三十分钟通常是足够为10–200µL 有机溶剂的容量。</li>
	<li>用大镊子在 Büchner 或真空瓶底部排列玻璃瓶, 用橡皮塞密封, 并将塑料油管连接到真空线上。夜间在轻度真空下疏散烧瓶, 清除残余有机物。</li>
	<li>覆盖脂膜整除数与惰性气体, 密封与密闭盖子, 并贮存在-20 °c, 用于在一个月或-80 °c, 以长期储存。</li>
	<li>制备含20毫米 naxhypo4 ph 6.8, 10% D2O 和洗涤剂 (+ 炸药) 的核磁共振样品缓冲器, 含20毫米 naxhypo4 ph 6.8, 10% D2o,0.34 毫米的 n-月桂-maltopyranoside。</li>
	<li>在带脂膜的玻璃瓶中加入 + 引爆缓冲器, 达到所需浓度, 在水浴中油脂实验30分钟, 交替超声波10分钟后进行目视检查。 注: 超声波可加热样品。在最终蛋白质样品重组前, 允许冷却至室温15分钟。</li>
	<li>使用1:1 混合物的超声波为所需浓度序列, 对脂洗涤剂胶束进行串联稀释。从125µM 脂浓度开始, 三重复将产生62.5 µM, 31.3 µM, 15.6 µM 脂在0.34 毫米的分布。</li>
	<li>分别制备蛋白质和添加剂的控制和参考核磁共振样品。添加蛋白质和添加剂, 每稀释脂在 + 引爆缓冲。 注: 15N NBB156, 蛋白质添加到最后浓度40µM 和补充2毫米氘 dithiothreitol, 以保持胱氨酸残留减少。</li>
	<li>将适当的样品体积转移到5毫米或3毫米核磁共振管 (见步骤5.4 中的说明)。</li>
	<li>在核磁共振仪器中手动加载样品或在自动化平台 (如 SampleCase 加载器) 中进行排列。</li>
	<li>利用标准脉冲程序获得1H 质子谱的复合核磁共振谱作为质量控制, 其次是 2D 1H-15N 相关谱作为横向松弛优化光谱学 (TROSY) 或异多量子相干性 (SOFAST-HMQC)33。 注: 使用3毫米核磁共振管需要64扫描 1H-15n TROSY (~ 3 小时) 或1h-15n SOFAST-HMQC (~ 90 分钟)。扫描数字为5毫米核磁共振管减半。</li>
	<li>经过处理的2D 核磁共振谱可以添加到卡拉资料库34中, 用于分析或供用户选择的其他软件。</li>
	<li>
通过注释2D 核磁共振共振为每个蛋白质状态的三个良好分散和分辨峰值分析软件。记录包含 NBB156 (图 5A) 的每个缓冲区条件中每个六峰值的峰值振幅。
	<ol>
<li>使用卡拉准备一批集成列表 (单击主菜单标题 "频谱 |安装批次列表 "...)所有的频谱收集和分析使用一个单一的峰值分配文件 (点击主菜单标题 "峰值 |导入 Peaklist "... 并选择用户定义的. 峰值文件, 其峰值为 6, 每个蛋白质状态为 3, 在内定义)。</li>
		<li>调整设置 ("集成商 |调峰模型 "...)X 宽 0.06 ppm 和 Y 宽 0.30 ppm (点击主菜单标题 "积分器 |调峰模型 "...)在将最终输出记录到两个菜单选择之前 (1。单击主菜单标题 "集成商 |集成批次列表 ") (2。单击主菜单标题 "峰值 |导出集成表 ") (图 5B)。 注: M21、V53 和 L105 残留物的骨干酰胺共振已被分配给 NBB156闭支撑状态。采用3D 核磁共振实验24, 将开放支撑状态分配给残余 G130 和 A141 的骨干酰胺共振和 W133 的氢质子侧链共振。</li>
	</ol>
</li>
	<li>通过将参考样品的三个开括号振幅平均, 并计算与这两个参照相关的正常化因子, 使样品直接与不同的磁铁或样品条件进行比较。使用规范化因子计算 NBB156共振的缩放振幅, 并将负振幅设置为零 (表 2)。 示例: 1) 比较不同磁铁的样品: 磁铁 A、NBB156+ 和磁铁 B、NBB156+。2) 洗涤剂比较: 0.34 毫米和1.7 毫米。</li>
	<li>计算每个共振的闭合或开放支撑的比例比例除以最小到最大振幅的所有光谱收集包含适当的参考自由 NBB156和完全开放大括号 NBB156在洗涤剂。</li>
	<li>将规范化的核磁共振振幅作为脂质浓度的函数进行绘制, 并比较 NBB156在不同条件下的比例 (图 5C)。 注: 另外, 对脂质浓度的闭合支撑构象的分数进行分析可以比较脂质结合 NBB156。我们更喜欢前者的分析, 因为它更快, 更好地报告的分数完全参与脂质。</li>
</ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIPK1+blocks+early+postnatal+lethality+mediated+by+caspase-8+and+RIPK3.">RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3.</a> Cell. 157 (5), 1189-1202 (2014).</li><li>Rickard, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIPK1+regulates+RIPK3-MLKL-driven+systemic+inflammation+and+emergency+hematopoiesis.">RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis.</a> Cell. 157 (5), 1175-1188 (2014).</li><li>Kaiser, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+3-mediated+necrosis+via+TRIF,+RIP3,+and+MLKL.">Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL.</a> Journal of Biological Chemistry. 288 (43), 31268-31279 (2013).</li><li>Upton, J. W., Kaiser, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAI+Another+Way:+Necroptotic+Control+of+Viral+Infection.">DAI Another Way: Necroptotic Control of Viral Infection.</a> Cell Host &amp; Microbe. 21 (3), 290-293 (2017).</li><li>Tanzer, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+signalling+is+tuned+by+phosphorylation+of+MLKL+residues+outside+the+pseudokinase+domain+activation+loop.">Necroptosis signalling is tuned by phosphorylation of MLKL residues outside the pseudokinase domain activation loop.</a> Biochemical Journal. 471 (2), 255-265 (2015).</li><li>Quarato, G., 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=Sequential+Engagement+of+Distinct+MLKL+Phosphatidylinositol-Binding+Sites+Executes+Necroptosis.">Sequential Engagement of Distinct MLKL Phosphatidylinositol-Binding Sites Executes Necroptosis.</a> Molecular Cell. 61 (4), 589-601 (2016).</li><li>Davies, K. 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我们提供的技术协议, 我们结合到牵连 MLKL 作为推定刽子手的等离子膜破裂24。除了破译调节 MLKL 介导的 necroptosis 的规管网络外, 这些技术还可以独立用于其他合适的生物系统。实际上, 这些技术是中等到低吞吐量的发现工具。
我们经常使用活细胞成像 NBB140-2 xfv-金星介导 necroptosis, 并诱导其他 MLKL 构造, 机械化解剖 MLKL 的调节 necroptosis 通过诱变和使?...

确定 necroptosis 的遗传成分有助于使用动物模型测试 necroptosis 在生理学和疾病1、2、3、4、5中的含义。RIPK3 或 MLKL 在小鼠中的击倒对发育和成人稳态的影响最小, 表明 necroptosis 对生命3,6不重要。此外, 某些物种不包含 RIPK3 或 MLKL 基因, 支持 necroptosis 在动物7,8的非必要的作用。另一方面, 在实验室诱发的各种病态的击倒动物模型揭示了 necroptosis 在炎症、先天免疫和病毒感染方面的重要作用,9,10,11,12。
Necroptosis 可以通过多种方式激活, 通过不同的先天免疫传感器发出信号, 所有这些都导致激活 RIPK31,13,14。活动 RIPK3 依次 phosphorylates 并激活 MLKL3、4、5、6、7、8、9、10 ,11,12,13,14,15,16,17,18。最受研究的, 也许是最复杂的方式, 导致激活的 RIPK3 涉及死亡受体结扎, 分叉的基础上的信号复合物的下游组成, 以诱导凋亡或 necroptosis1。Necroptosis 发生时, 通过 RIPK1 信号是青睐和结果参与 RIPK319,20。这一结果很容易被青睐的药理抑制或基因删除 caspase 8, 一个假定的内源性抑制剂 necroptosis, 保持 necroptosis 在海湾。RIPK1 绑定并激活 RIPK3。激活 necroptosis 的另一种方法是通过类似于 TLR3/TLR4 信号的像样的受体, 它通过域的适配器诱导的干扰素β (TRIF)21参与并激活 RIPK3。另一种死于 necroptosis 的方法是激活 DNA 传感器戴, 它直接参与并激活 RIPK322。
MLKL 是由一个 n-端螺旋束 (NB) 域和一个 C 终端 pseudokinase 域 (psKD) 组成的胞浆蛋白, 由一个调节支撑区3连接。在正常细胞中, MLKL 被发现在细胞质中, 它被认为是在一个不活跃的复杂与 RIPK314。激活 necroptosis 触发器 RIPK3 磷酸化的 MLKL 在 psKD 的活化回路, 并有可能在 NB 和支撑3,15,23的其他地点。磷酸化诱导 MLKL 的构象变化, 导致从 RIPK314的离解。不清楚的构象变化释放 psKD24的支撑。支撑, 其中包含2螺旋, 介导 MLKL 的齐聚三聚体通过 C 端螺旋25。支撑的 n-端螺旋抑制 NB 域, 这对于膜通透24,26是必不可少的。在分离的情况下, NB 域足以诱发等离子膜通透和 necroptosis16、24、27。在 MLKL (MLKL MEFs) 小鼠胚胎成纤维细胞中, necroptotic 活性为铌的重组。NB 是一种脂质结合领域, 优先从事磷脂磷酸肌醇45磷酸 (PIP2)。我们提出了 MLKL 活化的逐步机制, 其中大括号齐聚通过与 PIP2极性头组24的弱相互作用, 促进了 MLKL 到等离子膜的招募。在膜上, NB 对2的附加高亲和结合点进行了规范的曝光, 在非活动 MLKL 中由大括号屏蔽。总的来说, NB 与 PIP2的多重相互作用破坏了等离子膜的破裂, 尽管这些事件的分子机制尚未被阐明。
在这里, 我们说明了一些具体的方法来描述 MLKL 作为 necroptosis24的刽子手的功能。特别是, 我们专注于最微小的领域 MLKL, NB 和支撑 (NBB), 这是由支撑抑制, 可以通过强制二聚化激活, 以诱导等离子膜破裂和 necroptosis。我们描述了我们的诱导表达系统结合强迫药物诱导 FKBP 介导的二聚化的活细胞成像和电子显微手术细胞 necroptosis。此外, 我们还说明了我们的体外核磁共振分析的相互作用的 NBB 与 phosphatidylinositols (点子)。

<table border="0" cellpadding="0" cellspacing="0" style="width:1175px;" width="1174">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:429px;">Cloning and cell line generation</td>
			<td style="width:229px;"></td>
			<td style="width:323px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pRetroX-TRE3G</td>
			<td>Clontech</td>
			<td>631188</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tet-On transactivator plasmid</td>
			<td></td>
			<td></td>
			<td>Llambi et al., 2016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Embryonic Fibroblasts (MEFs) mlkl-/-</td>
			<td></td>
			<td></td>
			<td>Dillon et al., 2014</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin S Hydrochloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP2647100 CAS#3513-03-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell death quantification and live-cell microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxycycline</td>
			<td>Clontech</td>
			<td>631311 CAS# 24390-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B/B Homodimerizer AP20187</td>
			<td>Takara</td>
			<td>635059 CAS# 195514-80-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYTOX Green</td>
			<td>Thermo Fisher Scientific</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syto16</td>
			<td>Thermo Fisher Scientific</td>
			<td>S7578</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15N Ammonium Chloride</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>NLM-467-10 CAS# 12125-02-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterated DTT</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>DLM-2622-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deuterium Oxide</td>
			<td>Sigma Aldrich</td>
			<td>617385-1 CAS# 7789-20-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Dodecyl-&#946;-D-Maltopyranoside</td>
			<td>Anatrace</td>
			<td>D310 CAS# 69227-93-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-&#945;-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>840046X CAS# 383907-42-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (18:0 PI)</td>
			<td>Avanti Polar Lipids</td>
			<td>850143 CAS# 849412-67-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-dioleoyl-sn-glycero-3-phospho-(1&#39;-myo-inositol) (ammonium salt) (18:1)</td>
			<td>Avanti Polar Lipids</td>
			<td>850149 CAS# 799268-53-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Specialized Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IncuCyte FLR or ZOOM</td>
			<td>Essen BioScience, Inc.</td>
			<td></td>
			<td>Live-cell microscopy imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Helios NanoLab 660 DualBeam&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Electron microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IncuCyte 2011A Rev2 v20111.3.4288 (FLR)</td>
			<td>Essen BioScience, Inc.</td>
			<td>http://www.essenbioscience.com</td>
			<td style="width:193px;">Imaging analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FEI MAPS</td>
			<td>Thermo Fisher Scientific</td>
			<td>https://www.fei.com/software/maps/</td>
			<td style="width:193px;">EM analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TopSpin v3.2</td>
			<td>Bruker BioSpin</td>
			<td>http://www.bruker.com</td>
			<td style="width:193px;">NMR data collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CARA v1.9.1.7</td>
			<td></td>
			<td>http://cara.nmr.ch/&#160;</td>
			<td style="width:193px;">NMR data analysis</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:429px;">Slidebook</td>
			<td style="width:229px;">3i (Intelligent Imaging Innovations)</td>
			<td style="width:323px;">https://www.intelligent-imaging.com/slidebook</td>
			<td style="width:193px;">Confocal microscopy</td>
		</tr>
	</tbody>
</table>

characterization of mlkl-mediated plasma membrane rupture in necroptosis

Necroptosis 是由受体相互作用蛋白激酶 3 (RIPK3) 激活触发的程序化细胞死亡通路, phosphorylates 并激活混合谱系激酶样域 pseudokinase, MLKL, 破裂或 permeabilize 等离子膜.Necroptosis 是一种炎症通路, 涉及多种病理, 包括自身免疫、传染性和心血管疾病、中风、变性和癌症。在这里, 我们描述的协议, 可以用来表征 MLKL 作为刽子手的等离子膜破裂在 necroptosis。我们用常规和共聚焦荧光显微学的活细胞成像和固定细胞中的电子显微术来可视化细胞 necroptosis 的过程, 这一起揭示了 MLKL 从细胞质向血浆的再分布膜前诱导大孔的等离子体膜。我们采用体外核磁共振 (NMR) 分析方法, 用脂质来鉴定 MLKL 介导 necroptosis 的调节剂。基于这种方法, 我们确定了定量脂结合偏好和磷脂肌醇磷酸盐 (点子) 作为 MLKL 的关键粘合剂, 这是需要的血浆膜靶向和通透在 necroptosis。

通过最小截断 MLKL 构造, NBB140-2 xfv-金星的诱导表达, 可视化调节 necroptosis 在活细胞中的执行是可能的。该结构保持了诱导等离子膜通透的能力, 并通过 FKBP 盒 (2xFV) 的暗诱导齐聚而活化。我们观察和量化 necroptosis 的活细胞显微成像, 监测动力学 (每5分钟) 摄取的细胞不透水绿色荧光 DNA 结合染料 (图 6a)。这个诱导系统是非常健壮的,...

Watch this Scientific Journal Video about Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis at JoVE.com

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

我们报告的方法, MLKL 介导的等离子膜破裂在 necroptosis 包括常规和共聚焦活细胞显微成像, 扫描电镜, 核磁共振基脂结合。

Necroptosis MLKL 介导的血浆膜破裂特征

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

characterization-mlkl-mediated-plasma-membrane-rupture

Research

JoVE Journal

Biology

10.7K 次观看.  St. Jude Children's Research Hospital. 我们报告的方法, MLKL 介导的等离子膜破裂在 necroptosis 包括常规和共聚焦活细胞显微成像, 扫描电镜, 核磁共振基脂结合。

视频: Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

科研

生物学

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

测绘在细胞膜分子扩散，多目标跟踪（MTT）

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

娜功能分析<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt;细胞内的隔间使用质子打死选择器来表达它们的质膜

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...