我们感谢劳拉 Denti、瓦伦蒂娜 Senatore 和伦敦大学科学学院生物资源股的工作人员帮助小鼠饲养和卡米尔 Charoy 的技术支持。这项工作得到了医学研究理事会赠款 (MR/N011511/1) 的支持, Ruhrberg 和英国心脏基金会博士奖学金对 j.t. 傲慢 (FS/13/59/30649)。

所有的动物工作都是在英国内政部和机构动物福利和道德审查机构 (AWERB) 指导下进行的。
1. 鼠标准备
注: 对至少8周龄的成年小鼠进行实验, 年龄可达6月。为了演示在不同动物之间的每一个步骤的技术重现性和一致的时间, 每一个实验性的会议使用至少2和最多6只老鼠。为了评估突变对转基因小鼠的渗透诱导物质的影响, 理想情况下, 每项实验使用2变种小鼠和 2 littermate 控制。
<ol><li>24小时前刺激血管 hyperpermeability, 麻醉小鼠使用适当的吸入麻醉, 如异氟醚。使用3% 异氟醚进行麻醉诱导, 直到矫正反射丢失, 鼠标对外部刺激反应迟钝。使用1.5% 异氟醚进行麻醉维护 (确保鼠标有恒定的呼吸速率)。在麻醉下, 小心地用电动剃须刀剃掉每只老鼠的两侧, 避免皮肤受伤。 注: 麻醉是用来避免对动物造成压力, 并尽量减少剃须期间的运动, 这可能导致皮肤损伤。</li>
	<li>把剃光的老鼠送回家里的笼子里。对于雄性小鼠, 在麻醉恢复后将每个雄性置于一个单独的笼子里, 以防止打斗, 从而损害皮肤。对于雌性老鼠, 把他们送回他们的家庭笼子。 注: 如果在笼子里的雄性小鼠身上观察到打斗行为, 在手术前, 在实验前3天将它们分离成单独的笼子, 让潜在的皮肤损伤愈合。</li>
	<li>监视老鼠直到他们恢复知觉 (通常在几秒钟之内) 并且移动在笼子 (通常在1分钟之内)。</li>
</ol>2. 静脉注射埃文斯蓝染料
<ol><li>在层流柜中, 制备马来酸 pyrilamine 胺抑制剂 (4 µg/µL 0.9% 生理盐水) 和埃文斯蓝染料 (1% 瓦特/v 0.9% 生理盐水) 的分离无菌溶液。通过22µm 过滤器通过解决方案进行消毒。</li>
	<li>使用无菌的1毫升注射器与30克针腹腔注射每只老鼠与10µL pyrilamine 马来酸溶液/克的体重 (图 1A)。要执行注射, 首先, 把鼠标的背上, 然后倾斜, 使头部朝向地面, 腹部向上方向。在腹部的下象限内注射, 远离中线以避免撞击膀胱。 注意: 小鼠在8周至6月之间的体重通常介于15至30克之间. 马来酸 Pyrilamine 将抑制内源组胺的释放, 否则会促进血管渗漏, 独立于所测试的试剂。</li>
	<li>将鼠标放置在37摄氏度的热室中, 10 分钟促进血管舒张。或者, 使用一盏合适的热灯聚焦在尾部, 以促进血管舒张, 如果使用的热灯是允许的地方道德准则。</li>
	<li>一次将一只鼠标移动到鼠标抑制剂, 用70% 乙醇擦拭尾部以清洁注射区, 进一步促进血管舒张。</li>
	<li>使用无菌1毫升注射器与30G 针, 以管理100µL 埃文斯蓝色染料静脉注射通过尾静脉 (图 1B)。立即通过握住手指和拇指之间的尾巴来对注射部位施加压力, 以防止出血。 注意: 可以通过将灯光定向到注射区来改善尾静脉的可视化。关于对鼠标尾静脉进行静脉注射的进一步细节, 可在已发布的18号议定书中找到。</li>
	<li>允许染料循环30分钟。</li>
</ol>3. 刺激血管 Hyperpermeability
<ol><li>在层流柜中使用无菌溶液, 将感兴趣的渗透诱导剂稀释成浓度, 使最终剂量能在20µL 的体积内输送。 注: 在图 1所示的示例实验中-图 2中, VEGFA 用于刺激 PBS 中 2.5 ng/µL 浓度的血管 hyperpermeability, 总剂量为 50 ng, pbs 用作车辆控制。</li>
	<li>将渗透诱导剂的兴趣和车辆控制成2分不育300µL 注射器与31克针。加载足够的解决方案, 每三个注入20µL 的解决方案 (即准备60µL 的代理和车辆每只老鼠, 加上额外的体积, 以解释针的死容量)。</li>
	<li>麻醉第一只用异氟醚测试的老鼠 (3% 用于麻醉诱导, 直到矫正反射丢失, 鼠标对外部刺激反应迟钝, 1.5% 的麻醉维护, 确保老鼠有恒定的呼吸速率)。</li>
	<li>Intradermally 注入20µL 的渗透促进剂到鼠标的侧面 (图 1C)。确保针的角度与皮肤的一角。检查皮肤内凸起气泡的形成, 表明注射成功。重复2个额外的部位, 至少1厘米的皮注射液。</li>
	<li>转动鼠标暴露第二侧面和重复三个注射与控制车解决方案。在纸上记录每个注射部位的位置, 以方便他们识别随后收集的皮肤样品。 注意: 在这个阶段小心处理鼠标皮肤;例如, 在执行皮下注射时避免捏皮肤。</li>
	<li>将注入的鼠标返回到其主笼子并监视鼠标, 直到它恢复知觉 (通常在几秒钟内) 并在笼子周围移动。</li>
	<li>当第一个鼠标恢复时, 立即重复步骤3.3 和3.5 与第二个鼠标, 等等 (最多6小鼠每会话最大)。记录每个鼠标被注入的时间, 以调整进行后续步骤的时间。</li>
</ol>4. 在真皮内积累的埃文斯蓝的量化
<ol><li>在接受了皮下注射后, 用20分钟的宫颈脱位来剔除每只老鼠, 观察与注射小鼠相同的顺序。 注: 血管渗漏的持续时间可能根据所使用的渗透诱导剂而异, 因此, 皮下注射与剔除之间的时间可能需要优化。</li>
	<li>将每只被宰杀的老鼠放在背上, 把脚钉在一个裹着干净纸巾的软木板上 (图 1D)。</li>
	<li>使用钝剪刀, 使垂直切口大约 3-4 cm 从下腹部到死的老鼠的胸口。用镊子和手术刀, 从两侧的皮肤中梳理出真皮的内侧和埃文斯蓝堆积的部位 (图 1E)。针下松散的皮肤和清除周围地区的脂肪与手术刀。如果需要, 采取一个代表性的照片, 以证明埃文斯蓝色泄漏到皮肤的程度。</li>
	<li>使用镊子和手术刀, 消费的皮肤区域包括泄漏埃文斯蓝色染料为每个地点注入渗透诱导剂或车辆。 注: 注意在皮肤上同样大小的区域, 以确保在随后的步骤 4.7, 甲酰胺体积的类似部分将被每个皮肤样品吸附, 使提取的染料被稀释在类似的剩余的甲酰胺体积.步骤3.5 中记录的注入图将有助于确定要切除的区域。</li>
	<li>将每个样品放入1.5 毫升管中, 相应地贴上标签, 确保每个样品都放在管子的底部。将样品储存在-20 摄氏度, 直到进一步使用。如果立即处理, 请按照下列步骤操作。</li>
	<li>将打开的管子放在烤箱中或加热块的井中, 在55摄氏度的情况下, 在一夜之间干燥皮肤样品。</li>
	<li>要提取埃文斯蓝染料, 添加250µL 的去离子甲酰胺的样品在通风罩, 关闭管。确保所有的皮肤样品都覆盖的甲酰胺和孵化一夜之间的烤箱或加热块在55摄氏度。</li>
	<li>离心机样品在台式离心机以最大速度 (> 1万 x g) 为40分钟。</li>
	<li>在通风罩中, 将含有染料的上清液的100µL 从每个试样转移到一个透明的、平坦的底部96井板 (图 2A) 的一个独立井中。</li>
	<li>测量在620毫微米的峰值埃文斯蓝色吸光度与参考读数 740 nm 在分光光度计。 注: 620 nm 是埃文斯蓝吸光度的峰值, 而 740 nm 不被埃文斯蓝色吸收, 并充当参考波长。</li>
	<li>平均每三个相同的药剂或车辆的吸光度读数为每个鼠标, 以说明技术的变异性 (图 2B)。</li>
	<li>计算渗透诱导剂与车辆控制读数的折差 (图 2C)。</li>
</ol>

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作者没有利益冲突的声明。

自从171952年的第一个描述以来, 英哩法为研究者提供了一种相对快速、简单、可靠的方法来研究血管 hyperpermeability 的分子机制。例如, 英里分析被用来测试不同药剂的能力和/或效力, 以诱导 hyperpermeability19,20,21或测试 hyperpermeability 阻滞剂的功效8 ,22,23。作为另一个例子, 诱导血管通透性的药物已经在转基因小鼠及其 littermate 控制中进行了测试, 以确定配体诱导的特异受体和信号传感蛋白的要求 hyperpermeability答复10、11、12、13、14、15、20、23。从那以后, 已经使用了好几种改良版本的迈尔斯化验, 例如关于使用的示踪剂。因此, 埃文斯蓝已经取代了一些研究与荧光标记 dextrans 不同大小或微球11,13。
迈尔斯对其他检测血管 hyperpermeability 机制的研究的主要优点是它比较容易执行, 不需要昂贵的设备。此外, 作为一种体内技术, 这种检测模型在完整的血管背景下的血管渗漏, 而不是通过体外检测, 如通井流量测定或反式内皮电阻测量渗漏 (志愿者) 化验, 仅集中于内皮细胞单层。在体内测定血管 hyperpermeability 的方法可能不同于这里描述的英里分析, 利用不同的传递路线, hyperpermeability 诱导剂或分析不同部位的血管渗漏,例子通过全身交付代理通过尾巴静脉然后血管渗漏检查在肺或气管11,13,15。迈尔斯试验的一个局限性是, 某些制剂的皮下注射在原则上也可能影响血压和流量, 除了内皮屏障的破坏, 因此, 也间接影响血管通透性。然而, 这项检测最近被证明是测量 VEGF 诱发的血管通透性-独立于对全身血压的影响15。迈尔斯测定的另一个局限性是, 不同组织的血管床对渗透性促进剂的反应可能不同, 因此, 在皮肤中用英里数测定得出的结果可能不具代表性, 例如, 在肺或脑。
动物的年龄和体重可能会影响英里测定中所观察到的渗漏。为了尽量减少这些变量的影响, 研究人员应使用窝或类似大小和衰老的小鼠作为对照。当评估特定基因的小鼠突变体时, 应通过异型与异型育种策略和 wildtype 窝作为对照来生成小鼠。此外, 最好使用快速可逆的气体麻醉剂, 如异氟醚, 以避免血管收缩, 这已经描述了一些麻醉药物通过注射注射24,25。将埃文斯蓝染料注入小鼠侧尾静脉是本协议的关键步骤, 对实验和数据的质量有很大的影响。因此, 研究人员必须有能力执行尾静脉注射, 并可能需要事先的经验或实践之前开始的迈尔斯化验实验。另外, 研究人员也可以使用其他的静脉通路来提供埃文斯蓝色, 例如, 如果他们有经验的话, 可采用复古轨道注射。皮下注射渗透性促进溶液可以引起对皮肤的独立剂损伤。因此, 皮下注射不应超过20µl 的体积, 应始终进行在存在的组胺抑制剂和规范化的车辆控制注射。
许多研究人员已经用英里数法对 vegf 的成分进行了评估, 比如信号通路, 因为 vegf A 诱导的血管通透性会导致癌症患者16的腹水和视力损害在几个新生血管中的水肿眼部疾病7。因此, 我们和其他人已经使用了英里度分析来比较 vegf--一种诱导的小鼠血管渗漏, 基因修饰, 缺乏 vegf 的特定成分-信号级联12,13,14,15,20,23. 我们最近使用这种方法的研究表明, 主要的病理 VEGF--一种异构体, VEGF165, 通过 VEGFR2 和 NRP1 的复合物发出信号, NRP1 细胞质领域促进 SRC 家族激酶介导的内源性激酶的活化。唤起一个 hyperpermeability 的反应14。VEGFA 还促进血管的生长, 因此, 可以使用治疗, 以恢复血液流向缺血性组织, 如果其 hyperpermeability 活动可以特别抑制。研究表明, 引导 VEGF 的分子机制-血管内皮细胞对血管 hyperpermeability 的反应可能, 因此, 确定可选择性地操纵的途径, 以抑制病理 VEGF-诱发水肿疾病, 如癌症或缺血性眼病。迈尔斯的化验无疑将继续是一个有用的方法, 以支持这些和许多其他类型的功能研究, 以阐明分子调节因子和血管 hyperpermeability 的效应。

心血管系统的主要功能是使所有器官的循环和组织之间的气体、养分和废料的转移。血管有特定器官水平的基底通透性, 允许这种交流1。例如, 肾脏的血管是高度渗透性的, 而血脑屏障形成一个紧密的, 高度坚不可摧的界面2,3,4。形成血管内衬的内皮细胞在循环和底层组织之间提供了物理屏障, 并以特定器官的方式调节血管通透性。然而, 某些刺激导致内皮屏障的局部击穿, 以增加从循环的液体渗出到间质以上的基础水平1。例如, 在组织外伤、炎症、肿瘤、脓毒症、脑血管疾病或大脑和心脏等部位, 当组织缺血发生于中风或心肌梗塞时, 这些 hyperpermeability 被观察到, 分别5,6,7,8. 当长期升高时, hyperpermeability 会导致水肿, 进而导致组织损伤, 如眼部疾病中视力丧失9。因此, 对血管 hyperpermeability 反应进行建模, 对于了解增强内皮通透性的机制以及测试旨在抑制这种作用的药物的功效是可取的。
迈尔斯检测是一种公认的、常用的和相对简单的技术, 用于测量体内血管渗漏作为血管 hyperpermeability 的替代品。尽管它没有考虑到可能增加血管渗漏的复合因子, 如血压或血流等, 但通常认为英里法是一种可靠的方法来评估物质的渗透性调节活性, 并识别促进其活动的信号介质。因此, 迈尔斯的化验已经成为许多研究的一部分, 已经确定了血管 hyperpermeability 的调解人及其作用机制10,11,12,13, 14、15, 如血管内皮生长因子 VEGF-A 最初被确定为血管通透性因子 VPF16。
最初开发了几英里和英里的研究豚鼠血管通透性17, 他们的化验后来适应了使用小鼠, 这是目前的模式选择的有机体, 以阐明分子机制的血管通透性条例由于其精湛的适用性, 基因操纵。简言之, 埃文斯蓝染料是静脉注射到成年小鼠, 并允许循环30分钟 (图 1)。渗透诱导剂与车辆控制, 然后 intradermally 注射在多个站点上的反对侧翼的鼠标, 以诱发血管泄漏 (图 1)。因此, 白蛋白结合的埃文斯蓝染料 extravasates 和积累在真皮 (图 1)。在人道地剔除老鼠之后, 埃文斯蓝从真皮中提取出来, 并以测试物质的比率计算出的血管泄漏水平 (图 2)。

<table border="0" cellpadding="0" cellspacing="0" width="972">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:320px;">Pyrilamine maleate salt</td>
			<td style="width:141px;">Sigma-Aldrich</td>
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			<td style="width:167px;">Resuspend in PBS</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Deionised Formamide</td>
			<td>Sigma-Aldrich</td>
			<td>S4117</td>
			<td>Use in fume cupboard</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Microlance Needles 30g x 0.5&#34;</td>
			<td>BD&#160;</td>
			<td>305106</td>
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			<td height="40" style="height:40px;">Sterile Plastipak 1ml Luer</td>
			<td>BD&#160;</td>
			<td>309659</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:320px;">Sterile MicroFine syriinges 0.3 ml - 8 mm - 30G&#160;</td>
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			<td height="40" style="height:40px;">Evans blue</td>
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			<td>E2129</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Mouse restrainer</td>
			<td></td>
			<td></td>
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			<td height="40" style="height:40px;">Heat chamber</td>
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			<td></td>
			<td></td>
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			<td height="40" style="height:40px;">Hair clippers</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane</td>
			<td>Merial</td>
			<td>ap/drugs/220/96</td>
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			<td height="40" style="height:40px;">Scalpal&#160;</td>
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			<td height="40" style="height:40px;">Blunt scissor</td>
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			<td height="40" style="height:40px;">Forceps</td>
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			<td height="40" style="height:40px;">Eppendorf tubes</td>
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			<td height="40" style="height:40px;">Heatblock</td>
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			<td height="40" style="height:40px;">Recombinant Vascular Endothelial Growth Factor 165</td>
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</table>

evaluating vascular hyperpermeability-inducing agents in the skin with the miles assay

血管内皮在脊椎动物体内的主要功能是在血液和身体的每个组织之间起屏障作用, 从而使内皮细胞渗透到血球、血浆大分子和水中, 可以根据生理需要。在某些疾病中, 细胞因子和生长因子被释放, 靶向内皮屏障瞬时增加血管通透性;然而, 他们长期存在可能导致慢性血管 hyperpermeability, 从而组织损伤水肿。迈尔斯检测是一种体内技术, 允许研究人员通过代理测量血管渗漏来研究血管 hyperpermeability。在这里, 我们提供了一个详细的协议, 如何执行这个过程在鼠标, 这是最广泛使用的模型有机体, 研究哺乳动物的生理和病理学。该程序包括静脉注射埃文斯蓝染料标签的循环白蛋白后, 多次皮下注射渗透诱导剂和车辆控制解决方案, 以反对侧翼的鼠标。因此, 埃文斯蓝染料逐渐泄漏到真皮中, 它积累和可提取的量化作为渗透诱导剂相对于车辆的泄漏。英里分析可以在野生类型或转基因小鼠模型中进行, 并可与药物管理结合, 研究调节血管通透性的分子机制, 识别能够诱发或阻断的药物/靶点。hyperpermeability。

我们使用英里分析法评估血管内皮生长因子 A 在 wildtype C57/Bl6 小鼠中诱导的相对于车辆控制 (PBS) 的动脉泄漏的能力。在这里, 我们展示了一个典型的实验, 使用 wildtype 小鼠刺激与皮下注射20µL 溶液中含有 50 ng VEGF-a 在 pbs 或车辆 pbs 仅。在注射 VEGF 的皮肤样品中, 埃文斯蓝染料的渗漏明显增加--与 PBS 相比, 在原位可见 (图 1E) 和萃取后甲酰胺中的染料 (图 2A)。定量的多项实验表明, 50 的 VEGF-A 显着诱导更多的埃文斯蓝泄漏比 PBS 单独 (图 2B), 平均3倍增加的血管渗漏比车辆 (图 2C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57524/57524fig1.jpg"> 图 1: 在迈尔斯化验中诱导血管渗漏.在鼠标中执行英里检测时, 顺序步骤的示意图表示 (顶部面板) 和相应的图像 (底部面板)。(A) 马来酸 Pyrilamine 注射腹腔, 以防止内源组胺释放10至30分钟 (B) 静脉注射100µl 1% 埃文斯蓝到鼠尾静脉。(C) 30 分钟后, 血管渗漏是由制剂的皮下注射 (VEGF A; 绿色圆圈) 与车辆控制 (PBS; 白色圆圈) 引起的.(D、E)为了访问埃文斯蓝积累的网站, 老鼠被宰杀20分钟后, 皮下注射和皮肤两侧解剖和固定下来。腹腔内;静脉注射: 静脉;证件: 皮;刻度条, 1 厘米.<a href="/files/ftp_upload/57524/57524fig1large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57524/57524fig2.jpg"> 图 2: 在迈尔斯化验中量化血管渗漏.(A) 埃文斯蓝染料是从图 1中每种皮样中提取的皮肤样品中萃取的, 并装入一个96井板中, 用于吸光度法阅读。(B、C)通过绘制多项实验的吸光度读数的图形表示 (B) 渗透诱导剂 (VEGF A; 深绿色圆圈) 和车辆 (PBS; 灰色) 的归一化吸收值 (光密度、OD);圆圈), 或 (C) 代理与车辆 (误差条: SEM) 的 OD 变化。三种 PBS 和 VEGF 的吸光度读数 (a) 中的样品分别为白色或绿色圆圈, 以 (B、C) 显示。<a href="/files/ftp_upload/57524/57524fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay at JoVE.com

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

在这里, 我们提出了一个协议, 以测量的血管泄漏诱导的皮下渗透促进剂进入小鼠皮肤。该技术可用于确定分子促进或抑制血管渗漏的能力, 或研究调节血管通透性的分子机制。

用迈尔斯测定法评价皮肤血管 Hyperpermeability 诱导剂的含量

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

evaluating-vascular-hyperpermeability-inducing-agents-skin-with-miles

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.7K 次观看.  University College London. 在这里, 我们提出了一个协议, 以测量的血管泄漏诱导的皮下渗透促进剂进入小鼠皮肤。该技术可用于确定分子促进或抑制血管渗漏的能力, 或研究调节血管通透性的分子机制。

视频: Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.  (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

为持续给药Neuroactive代理Microinfusion系统的建设和植入。

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

科研

生物学

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量:贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

全安装胚胎肢体皮肤血管的免疫组织化学分析:一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

以血管内炎组织调节为重点, 利用肠系膜孤立动脉评价血管张力

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound

The retina within the eye is one of the most energy-demanding tissues in the body and thus requires high rates of oxygen delivery from a rich blood supply. The capillary lamina of the choroid lines the outer surface of the retina and is the dominating source of oxygen in most vertebrate retinas. However, this vascular bed is challenging to image with traditional optical techniques due to its position behind the highly light-absorbing retina. Here we describe a high-frequency ultrasound technique with subsequent flow-enhancement to image deep vascular beds (0.5-3 cm) of the eye with a high spatiotemporal resolution. This non-invasive method works well in species with nucleated red blood cells (non-mammalian and fetal animal models). It allows for the generation of non-invasive three-dimensional angiographies without the use of contrast agents, and it is independent of blood flow angles with a higher sensitivity than Doppler-based ultrasound imaging techniques.

We present a non-invasive ultrasound technique for generating three-dimensional angiographies in the eye without the use of contrast agents.

使用流量增强超声进行眼部深层血管成像

The retina within the eye is one of the most energy-demanding tissues in the body and thus requires high rates of oxygen delivery from a rich blood ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

通过粘附足迹测定法对细胞滚动中的分子粘附进行成像

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

从成年、幼年、幼虫和胚胎斑马鱼中分离心脏和血管平滑肌细胞以进行电生理研究

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...