作者要感谢罗杰斯实验室和白苹实验室的成员, 以及里德学院2018春季细胞生物学课程的学生, 他们为这个协议的发展做出了贡献。此外, 作者希望感谢丽思实验室的成员仔细阅读和编辑这篇手稿。该出版物的研究报告得到了国家科学基金会的支持, 716964 号奖项由 D.A.A. 和 A.R. 和里德学院生物系提供。

1.果蝇组织培养细胞的维护
<ol><li>保持 S2R+, S2R+:RLC-EGFP 和 S2:Fog-Myc 细胞在室温下, 最好介于20摄氏度和25摄氏度之间, 在盾和桑 M3 昆虫培养基补充10% 胎牛血清, 100 单位/毫升青霉素, 100 µg/毫升链霉素, 0.25 两性霉素 B 在一个密度不低于 5 x 105细胞/毫升。 注: 细胞密度降低导致死亡。</li>
	<li>快速解冻冷冻保存的小瓶细胞, 并将其转移到组织培养瓶中。从一个小的组织培养瓶 (12.5 厘米2或25厘米2) 的数量 4-5 毫升的细胞培养培养基, 直到文化建立和细胞死亡的迹象, 由于解冻已经消退。 注: 解冻后细胞死亡的迹象包括非黏附细胞, 它们缺乏平滑的、圆形的形状和组织培养介质中小的暗位细胞碎片, 表明坏死细胞。</li>
	<li>通过允许细胞不受干扰地生长数天 (4-8 d) 到100% 融合, 慢慢地扩大组织培养的大小, 在这些细胞之间几乎没有空隙, 它们开始互相堆积。转移所有细胞从这100% 汇合更小的 12.5 cm2或 25 cm2瓶到一个更大的组织培养瓶 (75 cm2) 补充与10毫升细胞培养培养基。 注意: 维持75厘米2瓶内的细胞将会产生足够的细胞用于大多数实验。</li>
	<li>通过吹打细胞培养基向上和向下, 每 3-4 维通过细胞, 利用它从组织培养塑料中去除任何松散黏附的细胞。把这个媒介, 现在包含悬浮细胞, 变成一个新鲜的瓶子。将细胞1:4 用新鲜细胞培养培养基稀释, 每段通过, 并根据需要重复。</li>
</ol>2.果蝇S2R+ 细胞的 rna 干扰治疗
注: 请参阅罗杰斯和罗杰斯6详细说明如何生产 dsRNA 适合昆虫文化。
<ol><li>将 S2R+ 细胞转移到6井或12井组织培养板中, 密度有利于生存, 不低于 5 x 105细胞/毫升, 使用完整的盾牌和桑 M3 细胞培养培养基进行 rna 干扰治疗。</li>
	<li>在组织培养罩中, 通过轻轻地倾斜6井或12井组织培养板, 并用吸管吸出细胞培养基, 去除旧的细胞培养培养基。用1毫升的新鲜细胞培养培养基, 通过吹打到每一个井中, 快速替换这个旧细胞培养基。 注意: 在交换细胞培养基之前, 细胞被允许松散地粘附 30-45 分钟, 这样可以在没有大量损失的情况下进行。</li>
	<li>在罗杰斯和罗杰斯6中, 添加10µg/毫升的 dsRNA, 通过吹打它直接进入每一个细胞的组织培养培养基每一个 rna 干扰条件。</li>
	<li>每天重复这一过程, 用1毫升新鲜培养基和10µg/毫升 dsRNA 每种干扰条件取代细胞培养培养基。 注: rna 干扰实验通常进行 7 d;然而, 确切的时间取决于蛋白质的周转和 dsRNA 的功效。3 d 的 rna 干扰治疗有效地耗尽了某些蛋白质的细胞, 而其他靶点需要更长的治疗6。</li>
</ol>3. 雾的生产和收获
<ol><li>扩大 S2:Fog-Myc 细胞的生长, 允许它们在 4-8 d 不受干扰的情况下生长, 在2毫升细胞培养培养基中获得近100% 个汇合的75厘米10瓶的细胞 (或者, 在150毫升的细胞培养基中, 2 厘米的20瓶) 到最大迈兹雾生产。</li>
	<li>添加50µL 100 毫米丘索4 (或100µL 100 毫米丘索4为150厘米2瓶) 到接近100% 汇合 75 cm2瓶为 metallothionine 促进剂的归纳。室温下孵育48小时。</li>
	<li>通过吹打从组织培养瓶中去除含雾的 c-myc 培养基, 并将其转移到15毫升或50毫升圆锥管上。离心机在 2500 x g在4°c 为 10-15 分钟清除细胞和细胞碎片的媒介。</li>
	<li>将清除的细胞介质转移到新的锥形管上, 并将其保持在冰上。分批工作, 转移5毫升清除介质到15毫升3000兆瓦集中器与再生纤维素膜和离心机它在 2500 x g在4°c 为30分钟, 集中媒介。 注: 另外, 可以使用容量较大的选矿厂 (50 毫升)。在浓度, 一个深色的介质, 这是丰富的雾, 将开始建立在底部的 V 形过滤器。</li>
	<li>将吸管尖端插入选矿厂的 "V" 底部, 并去除每轮离心之间的深色介质, 并将其转移到新鲜的1.7 毫升离心管。保持媒体在冰上。</li>
	<li>使用同一选矿厂, 重复步骤3.4 和 3.5, 通过删除深色的浓雾-c-myc 介质, 并取代它与更多的 precleared 介质, 直到介质已集中到 1/第十的原始体积 (即1 毫升从10毫升启动材料)。</li>
	<li>将浓雾在4摄氏度存储。 注: 贮存后, 一定要监测细菌污染情况, 这可能是由于贮存时间延长所致。</li>
	<li>通过混合10µL 浓雾-c-myc 培养基和10µL 的变性 SDS 缓冲液来进行 sds 页面形成裂解。运行 SDS 页凝胶在 35 ma 约 1 h. 将凝胶转移到硝化膜 (或另一种 PVDF 膜) 上, 在 120 mA 上进行1小时。使用任何商业上可用的 c-myc 抗体, 以检测 150 kDa 波段, 执行一个西方污点。 注: 在条件介质中的雾-c-myc 浓度可能每批次不同。为了进行检测, 没有必要确定雾的确切浓度, 但在进行实验之前, 应先测试每批的功效。</li>
	<li>以下步骤 3.1-3.2, 准备控制 S2-conditioned 媒体收集10毫升细胞培养基从近100% 汇合75厘米2瓶 S2 细胞 (或, 或者, 20 毫升从150厘米2瓶) 由吹打。将控制 S2 细胞培养基转化为新鲜的15毫升或50毫升圆锥管。</li>
	<li>以下步骤 3.4-3.7, 清除细胞和细胞碎片的裂解物通过离心 (在 2500 x g在4°c 每次30分钟), 然后转移清除的细胞裂解浓缩器和离心机他们集中控制介质分批1/第十原卷。 注: S2-conditioned 培养基的应用不会导致细胞收缩力的测定。</li>
	<li>将浓缩的 S2 控制介质存储在摄氏4摄氏度的几个月内。 注: 贮存后, 一定要监测污染情况。</li>
</ol>4. 刀豆蛋白涂层玻璃底菜的制备及 S2R+ 细胞的电镀
<ol><li>使10毫升的刀豆蛋白 a (a) 溶液溶解5毫克的10毫升的水, 最终浓度为0.5 毫克/毫升。整除这个解决方案成更方便的容量 (500 µL 到1毫升) 和存储它在-20 °c。</li>
	<li>准备35毫米玻璃底菜, 通过涂上每道菜的玻璃部分与200µL 的解决方案和孵化的菜肴约2分钟在室温下的组织培养罩。接下来, 删除所有的骗局一个解决方案 (可以保留和重用的未来实验), 并允许菜肴完全风干。 注: 菜肴可大量配制, 可在干燥处存放6月, 如实验室工作台抽屉。</li>
	<li>添加大约2毫升的新鲜细胞培养培养基, 每一个玻璃底菜。并用重悬 S2R+ 细胞通过吹打细胞培养基上下向上和向下的方法进行检测, 用它分离任何松散黏附的细胞。</li>
	<li>将悬浮 S2R+ 细胞转移到含有新鲜细胞培养基的35毫米玻璃底菜, 使它们在 50%-70% 之间汇合。在组织培养显微镜下检查细胞密度, 通过在培养基中悬浮的细胞中向上和向下聚焦, 得到细胞数的估计。 注意: 通过试验和错误, 可以达到 50%-70% 融合所需细胞的适当密度。</li>
	<li>允许细胞附着在镀膜玻璃底菜上 45-60 分钟。 注: 完全附着的 S2R+ 细胞出现 ' 煎蛋状, ' 扁平和相暗。</li>
</ol>5. 雾条件培养基的药效试验及细胞收缩力测定
<ol><li>如果需要, 通过 SDS 页和西方印迹确定 S2:Fog-Myc 稳定细胞系产生和集中的雾的屈服率。将含有10µL 的 SDS 样本缓冲区添加到10µL 浓雾中, 并将其煮沸5分钟. 遵循标准的 sds 页和西方的印迹协议和抗 c-myc 的印迹, 以确认其产生的雾。</li>
	<li>从步骤4.3 中的 S2R+ 细胞中取出所有的护盾, 然后轻轻吹打, 并小心地加回75µL 的新鲜盾和 M3, 以限制使用的雾条件介质的数量。</li>
	<li>添加75µL 的雾条件介质, 从步骤5.2 的玻璃底菜的玻璃部分, 使总体积到150µL, 在1:1 比例的雾条件的媒体, 以屏蔽和唱 M3 媒体。 注: 要完全覆盖典型玻璃底菜的玻璃部分, 需要 150-200 µL 的总容积;然而, 这将取决于使用的玻璃底菜的尺寸。</li>
	<li>用20X 或40X 倍度显微镜观察细胞的收缩力, 以观测整个细胞场。跟踪细胞的形态学, 寻找相暗和相光的交替模式梳理指示形状的变化。 注: 雾的应用通常导致 30%-50% 的 S2R+ 细胞进行细胞收缩, 并在10分钟后, 细胞开始放松, 恢复到他们光滑的边缘, 煎蛋样的形态。</li>
	<li>监控细胞收缩力的鲁棒性。如果细胞反应很强, 使用1:1 的雾条件介质与新鲜的盾牌和桑介质的比例, 减少雾条件介质的数量, 以保存浓雾-c-myc 介质, 为未来的实验。如果1:1 的比例不导致强健的细胞收缩, 使用未稀释的雾条件培养基;通常, 1:3 的比率产生足够的响应, 以成功地进行实验。 注: 一旦给定的浓雾条件介质中的雾条件介质与新鲜盾牌和桑培养基的适当配比, 则使用此比值进行所有其它实验。</li>
	<li>使用表达 GFP 标记的调节光链 (S2R+:Sqh-EGFP) 的稳定 S2R+ 细胞系, 监测非肌球蛋白 II 动力学在细胞收缩力试验中的动态。</li>
	<li>采用雾化条件培养基与新鲜盾构的比值, 从 5.2-5.5 步的 M3 培养基中去除细胞培养培养基, 并在玻璃底菜的玻璃板上加回新鲜的盾牌和桑培养基。在获取活细胞的图像序列的同时, 在预定比例灌注雾条件介质;在灌注过程中, 注意不要移动玻璃底菜。</li>
</ol>6. 收缩 S2R+ 细胞的固定和染色
<ol><li>固定 S2R+ 细胞使用10% 多聚甲醛溶液在 PEM 缓冲器 (100 毫米管, 1 毫米 EGTA, 1 毫米氯化镁2, pH 值 6.9)。 注: PEM 缓冲器通常是在2x 库存解决方案, 并可以存储几个月的4摄氏度。</li>
	<li>将细胞培养培养基从雾化处理的 S2R+ 细胞中取出吹打, 并立即用 1.5-2.0 毫升10% 多聚甲醛溶液取代。室温下孵育15分钟。 注: 固定细胞可贮存在4摄氏度的固定液中数天;然而, 重要的是, 最初的15分钟的固定是在室温下, 将细胞转移到4摄氏度之前进行。</li>
	<li>通过吹打去除10% 多聚甲醛溶液, 妥善处理此有机废料。将 1.5-2.0 毫升磷酸盐缓冲盐水 (PBS) 轻轻吹打到35毫米的玻璃底菜中冲洗细胞。删除旧 pbs 的吹打和更换 1.5-2.0 毫升的新鲜 pbs 2x, 以消除残留固定解决方案。 注意: 重要的是, 细胞不干, 从这一点上的协议。</li>
	<li>用大约200µL 5% 正常山羊血清稀释在 PBS 中的细胞, 在室温下用0.1% 非离子洗涤剂 (PBST) 补充20分钟。 注意: 通常, 阻塞解决方案包含与所引发的二级抗体相匹配的生物体的血清。</li>
	<li>在 PBST 中稀释它, 准备主要的抗体溶液。使用大约200µL 的体积, 完全覆盖玻璃底菜的玻璃部分。<ol>
<li>通过吹打删除阻塞解决方案, 并将主抗体溶液直接添加到菜的玻璃部分。在室温下孵育1小时, 或者在4摄氏度一夜之间孵化。</li>
		<li>在4摄氏度的一夜之间孵化时, 用 parafilm 包裹35毫米的玻璃底菜, 以防止蒸发。</li>
	</ol>
</li>
	<li>去除主要抗体溶液通过吹打和冲洗细胞通过吹打 1.5-2.0 毫升新鲜 PBS 到35毫米玻璃底菜。删除 pbs 的吹打和更换 1.5-2.0 毫升的新鲜 PBS 2x 更多。</li>
	<li>通过稀释 PBST, 制备二次抗体的溶液。200µL 的体积足以覆盖盘子的玻璃部分。<ol>
<li>将二次抗体溶液直接添加到盘子的玻璃部分。用二级抗体溶液在室温下或一夜4摄氏度孵育细胞1小时。</li>
		<li>如果一夜之间孵出35毫米的盘子, parafilm, 以防止蒸发。</li>
	</ol>
</li>
	<li>去除二次抗体溶液通过吹打和冲洗细胞与 PBS 由吹打 1.5-2.0 毫升新鲜 pbs 对菜。删除旧 PBS, 并根据相同的过程更换2x。</li>
	<li>通过添加足够的防褪色荧光安装介质来保护固定的和染色的细胞, 以覆盖玻璃底部的35毫米盘子的玻璃部分。</li>
	<li>将样品存放在摄氏4摄氏度, 远离光线。 注意: 即使正确存储, 荧光信号也会在数月内慢慢衰减。为了取得最佳效果, 在一周内图像。</li>
</ol>7. 细胞收缩力测定的影像学和量化
<ol><li>图像固定 S2R+ 细胞使用标准的倒置光显微镜与相对比, DIC, 或荧光成像能力。使用20X 或40X 放大倍数, 捕获 10-30 不重叠图像域的细胞密度, 取决于细胞浓度, 目标是获取200或更多的细胞每一个条件的图像。</li>
	<li>使用标准单元格计数器, 计数并记录每个字段捕获的收缩 (相暗、bonneted 单元格) 和 noncontracted 单元格的数量。 注意: 最好有两个研究人员对每个领域的收缩和 noncontracted 细胞独立计数, 或者, 同样的研究员可以计算相同的细胞图像领域三倍, 允许平均数量的收缩和要计算每个字段的 noncontracted 单元格。</li>
	<li>按照步骤4.2 中的描述, 每一个实验条件下准备几个镀膜玻璃底菜。吸管向上和向下的细胞培养培养基, 以赶走任何松散黏附 S2R+:Sqh-EGFP 细胞和板块, 使他们是 50%-70% 汇合在35毫米玻璃底菜。允许单元格附加 30-45 分钟。</li>
	<li>图像 S2R+:Sqh-EGFP 细胞使用旋转圆盘, 扫频场共聚焦显微镜, 或通过光片显微镜使用 60X-100X 放大倍数。除去所有的盾牌, 并从含有附着 S2R+:Sqh-EGFP 细胞的玻璃底菜中 M3。<ol>
<li>灌注从步骤 5.2-5.5 直接到玻璃底盘的玻璃部分, 用吸管, 小心地不干扰菜的位置在目标, 被预定的比例的雾-c-myc 被适应的媒介到新鲜的盾和唱歌 M3 媒介。获取 12-15 分钟的图像, 以捕捉 S2R+:Sqh-EGFP 细胞的启动和完全收缩。 注: 如果染色的非肌球蛋白或肌球蛋白-pRLC, 形成一个紧密的肌球蛋白环在细胞表面也表明收缩力。如果成像 S2R+:Sqh-EGFP 细胞活着, EGFP 标记的 RLC 将形成类似的紧环 (见图 3), 表明收缩力。发生的形状更改将导致单元格 "阀盖", 占用3D 形状;显微镜焦距的平面可能需要调整以跟踪这个细胞的形状变化。在控制 rna 干扰条件下, 通常, 30%-50% 的细胞在添加雾时会受到收缩。请注意, 某些干扰条件可能会导致在没有雾的情况下收缩。</li>
	</ol>
</li>
</ol>

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作者声明他们没有竞争的财政利益。

在这里, 我们提出了一个详细的协议, 以细胞为基础的收缩力试验使用果蝇组织培养细胞线 (S2R+ 细胞), 经历非肌球蛋白 II 收缩作为回应雾信号。这种检测方法对于研究雾化通路以及调节非肌球蛋白 II 收缩力的机制是有用的。
细胞培养注意事项: S2R+ 细胞的维持条件对于从该检测中获得可靠数据至关重要。当计划执行收缩力试验时, 最好保持 S2R+ 细胞在盾牌和 M3 昆虫培养基。虽然 S2R+ 细胞可以在其他昆虫细胞培养媒介 (如SF900 或施耐德的昆虫培养基) 中茁壮成长, 但它们往往会失去对雾的反应。S2R+ 细胞有很高的通道数, 一般超过 20-25 通道, 开始失去对 rna 干扰的敏感性。最好使用早期通道细胞进行所有实验。干扰损耗实验的另一个重要方面是选择适当的控制。常见的阴性控制包括 dsRNA 靶向 EGFP 或 pBlueScript, 两者都与苍蝇基因组没有同源性。靶向蛋白在雾通路 (洛, 韩等), 其中, 当耗尽, 防止激活非肌球蛋白收缩力, 也是有用的控制。S2:Fog-Myc 细胞可以保持在替代细胞培养培养基, 如 SF900 补充100单位/毫升青霉素, 100 µg/毫升链霉素, 0.25 两性霉素 b. 注意在 SF900 培养细胞时不需要使用血清。细胞密度也是果蝇组织培养细胞各方面的重要组成部分。与哺乳动物组织培养细胞不同,果蝇衍生的组织培养细胞在较高的细胞密度下生长得最好 (密度不低于 5 x 105细胞/毫升)。然而, 当进行这项化验时, 关键的是, 细胞没有被镀在一个涂层玻璃底菜超过80% 汇合, 因为定量的收缩与非收缩细胞将变得极其乏味。
细胞收缩力测定的关键方面和替代方法: 雾的生产, 触发非肌球蛋白 II 收缩力的配体, 是这项试验的一个关键组成部分。雾基因对730种氨基酸的蛋白质进行编码, 预测的分子量为 78 kDa26。Hydropathy 分析显示, 在雾的氨基端有12疏水性残留物, 可以作为分泌信号序列。此外, 编码序列还包含多个站点的潜在 n-和 O 链接糖基化, 进一步建议雾是分泌蛋白26。为支持这一点, 雾被局部化为分泌囊泡在假定表皮细胞接受顶端收缩16。在培养基中加入硫酸铜后, 产生了雾-c-myc 的表达, 而抗雾或 c-myc 抗体则从诱导培养培养基中提取出 150 kD 蛋白, 而不是从 S2 细胞的诱导培养基中提取。这一分子量高于预测的 80 kD 的雾分子量, 表明 S2 细胞分泌机械在胞吐作用之前可能 glycosylate 蛋白质。由于雾净化的潜在变异, 在所有实验中使用同一批雾化条件介质是可取的。制作大量浓雾--c-myc 介质将有助于保持整个实验的一致性。
这种化验方法的成功还取决于有信心地识别已经历收缩的细胞。虽然收缩细胞可以通过荧光显微镜观察, 通过染色肌动蛋白或非肌球蛋白 II, 最可靠的方法来识别和量化收缩细胞是通过相对比或 DIC 显微镜。在大多数标准光学显微镜上, 使用 20X-40X 放大倍数可以实现精确计数。尽管这里写的协议使用的是玻璃底菜, 但也可以使用标准的1.5 玻璃盖玻片涂上 A。添加雾可以在35毫米的组织培养上的盖玻片放置在 parafilm, 以限制每种化验所用的雾量。固定质量是检测的一个重要组成部分, 因为固定细胞的不良会导致误报。使用新鲜的固定液和确保细胞不干燥一旦固定将导致更可靠的结果。最后, 对大量的单元数进行计数是很重要的。通常情况下, 只有 30%-50% 的未经治疗或控制的 rna 治疗细胞收缩后, 雾的灌注。然而, 在 S2R+ 细胞中出现了基础性的收缩, 因此需要大量的细胞来确保收缩细胞的分数的任何变化都是由治疗引起的。此外, 在调节非肌球蛋白 II 收缩的部分蛋白质的消耗可能导致超收缩性, 即使在没有雾。这里提供的数据来自3600多个细胞, 在三个连续的 rna 干扰处理中, 使用同一批次的雾条件介质进行计数。
细胞收缩力测定的应用: 这种收缩力分析, 当加上 rna 筛选方法, 提供了一个强大的系统来研究细胞信号, 形态发生和细胞收缩力。以前, 它已经被用来识别两个雾共受体之一21。通过在 S2R+ 细胞中进行 rna 干扰, 对138克蛋白偶联受体 (受体) 进行靶向筛查, 并根据此处介绍的协议, 测定其对雾反应的能力。在138受体中, 一个先前 uncharacterized 的基因, 现在被称为薄雾, 被发现。对薄雾功能的进一步研究表明, S2R+ 细胞不仅需要雾诱导的细胞收缩力, 而且肠在果蝇21的发育中也是必不可少的。此外, 这一分析被用来证明 Ric-8, 一个非规范的全球环境基金, 也是雾信号通路27中的一个组成部分。一系列的上位 rna 干扰实验和收缩力试验表明, Ric-8 与 Gα12/13亚基 Cta 和功能, 以本地化它在细胞内, 这是关键的雾诱导细胞收缩力31.
果蝇组织培养非常适合初学者, 因为细胞在室温下很容易保持, 不需要 CO2或缓冲细胞培养培养基, 只要保持适当的细胞培养密度, 就能强健。细胞收缩试验成功地由实验室部分进行的本科生细胞生物学课程, 在那里的学生几乎没有任何经验, 培养细胞或显微镜。这里提出的细胞收缩力检测是一个强大的, 细胞基础的工具, 可用于基因发现, 或询问雾信号通路, 帮助我们更好地了解发展过程, 如顶端收缩和非肌球蛋白 II. 收缩, 一般。

用遗传模型生物体进行胚胎发生的研究对我们理解细胞如何组装成组织是非常宝贵的。对果蝇的研究, 尤其导致了关键基因和生物学原理的鉴定, 它引导生物体的形态发生和发育, 从受精到成年1,2,3. 在与果蝇进行的遗传学研究的同时, 从果蝇组织获得的培养细胞系也成为一个强有力的系统来处理各种分子和细胞生物学问题4,5,6.果蝇组织培养细胞对维持的要求最低, 因为它们在室温下培养, 没有 CO2。因此, 它们是适合高分辨率成像, 他们有高度的敏感性基因抑制的 rna 干扰6,7。许多团体使用果蝇组织培养细胞作为一种工具来发现相关基因定义细胞形态, 骨架动力学, 生存能力, 吞噬功能, 信号转导通路4,8, 9,10,11。当作为一个模型, 与整个动物, 养殖果蝇细胞线提供了一套非常互补的方法, 加快识别重要的分子的发展, 并提供一个框架, 其中确定他们的机械角色12。在这里, 我们描述了一种基于细胞的检测方法, 研究通过折叠-肠 (雾) 通路13,14触发顶端收缩的信号通路。这种基于细胞的收缩力检测方法允许研究人员研究雾化信号通路和调节非肌球蛋白 II 收缩力的分子机制。
早期果蝇胚胎的肠已被研究多年, 作为一种遗传模型的上皮形态发生和细胞从上皮细胞过渡到间充质的身份。肠的关键事件之一是在15、16、17、18形状的胚胎腹侧中线形成的上皮细胞子集的形态发生。这种简单的细胞形态变化导致推定的胚层细胞内化, 并由非肌球蛋白 II 的运动活动驱动, 收缩肌动蛋白网络16,19,20。数十年的基因研究已经确定了这一通路的分子成分, 并按顺序将它们排列在下面: 1) 从腹中线上皮细胞的顶端区域分泌出雾;2) 雾与 g 蛋白耦合的共受体、薄雾和烟雾结合, 并通过含有 Gα12/13亚单位铁丝网 (Cta) 的 heterotrimeric g 蛋白复合物发出信号, 这是由非规范的全球环境基金 Ric-8 陪护的;3) Cta 激活鸟嘌呤核苷酸交换因子 RhoGEF2, 进而激活小 G 蛋白 Rho1;4) Rho1 激活蛋白激酶 (韩);5) 韩国通过调节光链 (RLC) 的磷酸化来激活顶端域的非肌球蛋白 II 收缩力, 从而产生顶端收缩 (图 1)15、21、22 ,23,24,25,26,27,28,29。这些成分中的突变会干扰正常的肠和其他形态发生运动, 包括翼盘和唾液腺的形成, 表明这条通路在果蝇的几个阶段被使用。胚胎发生30,31,32。雾通路是最受研究的上皮整形模型之一, 并提供了重要的洞察力如何从基因转录调控细胞细胞运动14,15 的组织级形态发生 ,21。
我们开发了一种基于细胞的收缩力检测方法, 概括了在开发飞胚17中观察到的雾下游的许多细胞反应。我们设计了一个稳定的 S2 细胞线, 表示在它的 C 总站标记的雾在一个可诱导的 metallothionine 启动子的控制下, 可以收获后, 添加硫酸铜 (丘索4) 到培养基。当雾条件的媒介被应用 ectopically 对 S2 受体 + (S2R+) 细胞, 这是 S2 细胞的亚系, 区别于它们的受体的差异表达, 如卷曲和整合素亚基, 细胞经历重组的细胞骨架高度令人联想到顶端收缩12,17,27,33。这些变化可以通过相对比显微镜观察到, 在这种情况下, 雾处理导致了相色褶边的出现, 表明在非肌球蛋白 II 收缩力的径向增加, 或通过荧光显微镜在那里雾处理导致表达 EGFP 标记 RLC34的细胞中非肌球蛋白 II. 型环的形成。这些环含有肌球蛋白磷酸化调节光链 (pRLC) 可见通过染色23,34,35。这种雾引起的反应需要 Cta, RhoGEF2, 洛, 和韩国;因此, 利用重组的 S2R+ 和细胞, 我们建立了一种方法来研究雾诱导的收缩在组织-文化为基础的系统24,25,34。

<table border="0" cellpadding="0" cellspacing="0" style="width:1159px;" width="1157">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">S2R+ cells</td>
			<td style="width:223px;">Drosophila Genomics Resorce Center&#160;</td>
			<td style="width:123px;">150</td>
			<td style="width:496px;">cell culture line, undergoes apical constriciton</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">S2:Fog-myc cells</td>
			<td style="width:223px;">Drosophila Genomics Resorce Center&#160;</td>
			<td>218</td>
			<td>cell culture line, produces Fog-myc under pMT promoter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">S2R+ :Sqh-EGFP cells</td>
			<td style="width:223px;">Drosophila Genomics Resorce Center&#160;</td>
			<td>196</td>
			<td>cell culture line, stably expresses Sqh(RLC of non-muscle myosin II) tagged with EGFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Shield and Sang M3</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:123px;">S3652</td>
			<td>cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">SF900 insect medium</td>
			<td style="width:223px;">Thermofisher Scientific</td>
			<td>12658019</td>
			<td>alternative cell cutlure medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Schneider&#8217;s insect medium</td>
			<td style="width:223px;">Thermofisher Scientific</td>
			<td>21720024</td>
			<td>alternative cell cutlure medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Fetal Bovine Serum</td>
			<td style="width:223px;">Thermofisher Scientific</td>
			<td>16000044</td>
			<td>media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic-Antimycotic (100X)</td>
			<td style="width:223px;">Thermofisher Scientific</td>
			<td>15240112</td>
			<td>antimicrobial/antifugal media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Concanavalin A</td>
			<td style="width:223px;">MP Biomedicals</td>
			<td style="width:123px;">150710</td>
			<td>used to coat dishes and coverslip for cellular adhesion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Glass bottom plates</td>
			<td style="width:223px;">Maktek</td>
			<td style="width:123px;">P35G-1.5-10</td>
			<td>used for assay and for fixing and staining cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Glass cover slips</td>
			<td style="width:223px;">Corning</td>
			<td style="width:123px;">2940-225</td>
			<td>alternative to glass bottom dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Protein concentrators</td>
			<td style="width:223px;">Millipore</td>
			<td style="width:123px;">UFC903008</td>
			<td>30,000 molecular weight cutoff, used to concentrate media</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:317px;">25 cm2, 75 cm2, 150 cm2 tissue culture flasks</td>
			<td style="width:223px;">Genessee</td>
			<td style="width:123px;">25-204,25-206,25-208,25-210</td>
			<td>used to culture cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">6-well and 12-well tissue culture dishes</td>
			<td style="width:223px;">Genessee</td>
			<td style="width:123px;">25-105, 25-106</td>
			<td>used to culture cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Flourescent mounting medium</td>
			<td style="width:223px;">Dako</td>
			<td style="width:123px;">S3023</td>
			<td>to preserve fixed cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">32% Paraformaldehyde</td>
			<td style="width:223px;">EMS</td>
			<td style="width:123px;">15714-S</td>
			<td>used to fix cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Pipes</td>
			<td style="width:223px;">EMS</td>
			<td style="width:123px;">19240</td>
			<td>used in PEM buffer for cell fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Anti-Myc Antibody</td>
			<td style="width:223px;">Drosophila Hybirdoma Bank</td>
			<td style="width:123px;">cat# 9E10</td>
			<td>used to dectect Fog-Myc&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">PhosphoSerine-19 antibody</td>
			<td style="width:223px;">Cell Signaling</td>
			<td style="width:123px;">3671</td>
			<td>antibody against phosphorylated regulatory light chain (RLC) serine 19</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">488-Phalloidin, 568-Phalloidin</td>
			<td style="width:223px;">Thermofisher Scientific</td>
			<td style="width:123px;">A12379, A12380</td>
			<td>to stain for f-actin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hawk-VT multi-point array scanning confocal system&#160;</td>
			<td style="width:223px;">VisiTech International</td>
			<td style="width:123px;"></td>
			<td>Used for live cell imaging of S2R+:Sqh cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Eclipse TE300 Inverted microscope</td>
			<td>Nikon</td>
			<td style="width:123px;"></td>
			<td>Used for live cell imaging of S2R+:Sqh cells</td>
		</tr>
	</tbody>
</table>

a cell-based assay to investigate non-muscle myosin ii contractility via the folded-gastrulation signaling pathway in drosophila s2r+ cells

我们开发了一种基于细胞的检测方法, 使用果蝇细胞概括顶端收缩由折叠的肠 (雾), 分泌上皮 morphogen 发起。在这项试验中, 雾被用作一种激动剂, 通过信号级联, 其中包括 G 蛋白耦合受体 (薄雾), Gα12/13蛋白 (铁丝网/Cta), 和一个 PDZ 域包含鸟嘌呤核苷酸交换因子 (RhoGEF2) 激活。雾信号导致肌动蛋白细胞骨架的迅速和戏剧性的重组形成一个收缩的钱包字符串。可溶性雾是从一个稳定的细胞线收集和应用 ectopically 到 S2R+ 细胞, 导致形态学变化, 如顶端收缩, 一个过程中观察到的发展过程, 如肠。这种检测方法适于高通量筛选, 而且利用 rna 干扰, 可以帮助识别这一途径中的其他基因。

S2 和 S2:Fog-Myc 细胞生长到接近汇合条件在一个 150 cm2组织培养瓶和被治疗与 25-75 µM 丘索4在 24-48 小时期间导致的雾的表达 (图 2)。S2 细胞 (图 2A) 和 S2:Fog-Myc 细胞 (图 2B) 中的样本被去除, 并由西方的印迹监测了该生产的雾-c-myc。正如我们所预料的那样, 在任何条件下, 我们都无法检测到从 S2 细胞中收获的介质中的雾-c-myc。然而, 我们确实检测到从 S2:Fog-Myc 稳定细胞线采集的介质中的雾, 早在24小时后诱导与75µM 丘索4。S2R+ 细胞附着在一个涂层的玻璃底菜上, 表达了 EGFP 标记的调节光链 (GFP-RLC) 的非肌球蛋白 II (图 3A和3B)。在有雾条件的介质的灌注过程中, 在装有 100 x/1.4 NA 物镜的倒置显微镜下进行活细胞成像。大约5分钟后处理与雾-c-myc, RLC 形成的圆环指示非肌球蛋白 II 收缩。这些环可以通过染色 S2R+ 细胞来观察, 它的抗体与人体肌球蛋白 RLC Ser19 周围残留的合成磷酸相对应 (图 4A和4B)。这种抗体会与果蝇的 RLC 相互反应。
在这里, 我们有一个代表性的例子, 细胞收缩力检测后, 7 天的治疗细胞与控制 rna 干扰和 dsRNA 对 Rho1 (图 5A - 5E)。S2R+ 细胞被镀在一层玻璃底碟上, 用雾条件 (+ 雾) 介质或控制介质 (雾) 处理。在固定后, 收缩细胞的数量被量化。相密集的褶边表明收缩在控制耗尽的细胞被雾条件的媒介处理 (图 5B) 突出。在图 5C中, 用 S2-conditioned 培养基处理的细胞是光滑、煎蛋样形态的典型例子。请注意, 在雾信号通路中, 和在胞质分裂中, 它扮演着一个角色;因此, 在细胞体积和倍性的增加中, dna 耗尽的细胞往往无法正确地分化。用雾化条件的培养基治疗控制衰竭细胞典型地导致 30%-50% 的细胞在收缩, 而我们没有观察到在衰竭细胞的坚固收缩。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58325/58325fig1.jpg"> 图 1:假定的雾信号通路.在没有雾的情况下, Gα12/13亚基, 铁丝网 (Cta), 连同其具有约束力的合作伙伴 gυ和 Gβ, 是不活跃的, 并与共同受体雾和烟雾相关。Ric-8 伴侣 Cta 和调节其亚细胞定位。在雾的捆绑, Cta 电离从 gυ和 Gβ新兵 RhoGEF2 到细胞膜那里它催化 GDP 的交换 GTP 在小 GTPase Rho1。GTP Rho1, 现在活跃, 激活蛋白激酶 (韩国), phosphorylates 的 RLC 的非肌球蛋白, 导致其活化和随后的细胞收缩力。<a href="https://www.jove.com/files/ftp_upload/58325/58325fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58325/58325fig2.jpg"> 图 2: 产生雾的时间过程.这些面板显示从近100% 个 (a) 控制 S2 细胞的汇合瓶或 (B) S2:Fog-Myc 稳定细胞中收获的细胞培养培养基。细胞在 24-48 小时内用 25-75 µM 丘索4治疗。收集并制备了细胞培养培养基, 用于 SDS 页和西方印迹。用抗 c-myc 抗体检测出了该病毒的 c-myc。<a href="https://www.jove.com/files/ftp_upload/58325/58325fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58325/58325fig3.jpg"> 图 3: 雾诱导收缩的活细胞成像.用扫描场共聚焦显微镜在雾条件介质的灌注过程中, 对表达 EGFP 标记 RLC 的非肌球蛋白 II. 的稳定 S2R+ 细胞系进行了成像。时间以分钟和秒显示。(A) 此面板显示了在盖玻片之前的0:00 时间内, 靠近细胞-界面的焦平面的图像. (B) 这个小组显示了一个时间序列 (0:30-10:00) 在靠近细胞顶部的焦平面上拍摄的图像。白色箭头表示重组的非肌肉肌球蛋白 II 表示, EGFP-RLC 到收缩环期间收缩。刻度条为10µm.<a href="https://www.jove.com/files/ftp_upload/58325/58325fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58325/58325fig4.jpg"> 图 4: 在本地化的诱导下, 磷酸化-RLC 为收缩环.S2R+ 细胞是固定和染色的肌动蛋白使用罗丹明 (红色), 磷酸化 RLC 使用 phosphoserine-19 抗体 (绿色), 和 DAPI (DNA, 蓝色), 后治疗与 (a) 控制或 (B) 雾-c-myc 条件的媒体。请注意, 加入雾-c-myc 后, 磷酸化 RLC 成环的重组.<a href="https://www.jove.com/files/ftp_upload/58325/58325fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58325/58325fig5.jpg"> 图 5: 对雾诱导的细胞收缩力测定的代表性量化.S2R+ 细胞 (A和B) 控制 rna 干扰或 (C和D) dsRNA 对小 GTPase Rho1 7 天。在此之后, 这些细胞被镀在一层玻璃底的盘子上, 并用 (A和C) 控制介质或 (B和D) 的雾-c-myc 条件的媒介进行治疗。刻度条代表10µm. (E) 散点图表示每个条件下的收缩细胞分数的平均和95% 置信区间。单个点代表每个视场中的收缩细胞的分数 (每个字段的 10-120 个单元格), 括号中的数字表示在三个单独的收缩和非收缩细胞中计数的细胞总数。rna 干扰实验。星号表示在 Tukey 的多比较后分析后, 由单向方差计算 (p < 0.0001) 确定的干扰和雾化处理条件之间的统计学意义差异。请注意, 对照治疗的 Rho1 rna 处理的样品和用雾-c-myc 条件的培养基之间没有统计学意义上的差异 (生理盐水)。<a href="https://www.jove.com/files/ftp_upload/58325/58325fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells at JoVE.com

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

这里我们描述了一种使用果蝇S2R+ 细胞的收缩力测定方法。外源配体, 折叠肠 (雾) 的应用, 导致了雾信号通路和细胞收缩力的活化。该方法可用于研究雾信号通路中细胞收缩蛋白的调节。

果蝇S2R+ 细胞折叠-肠信号通路检测非肌球蛋白 II . 收缩力的细胞基础研究

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...

a-cell-based-assay-to-investigate-non-muscle-myosin-ii-contractility

Research

JoVE Journal

Developmental Biology

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

7.4K 次观看.  The University of North Carolina at Chapel Hill. 这里我们描述了一种使用果蝇S2R+ 细胞的收缩力测定方法。外源配体, 折叠肠 (雾) 的应用, 导致了雾信号通路和细胞收缩力的活化。该方法可用于研究雾信号通路中细胞收缩蛋白的调节。

视频: A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

科研

发育生物学

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

建立有限元模型，研究斑马鱼下颌生物力学

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

HUVEC 管形成测定评估天然产物对血管生成的影响

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...