我们感谢下列协助畜牧和收集数据的人: d. Tegtmeier、c. Fossen、波斯富、m. 麦克威廉姆斯、l. Bertola、斯图尔特、n 哈尼和 t Knavel;和 m. Merces 协助解剖。我们还要感谢 m. 麦克法登、哈洛和塔隆加动物园提高低压阿尔宾娜和 g. Marantelli 以提高 P. corroboree。我们感谢 f. Pasmans, a. 马特尔关于细胞凋亡化验的建议, 康斯坦丁, Kladnik 和韦伯的帮助与隧洞化验, t Emeto 和 W. Weßels 的帮助与协议和试剂盒 caspase 3/7 化验。此手稿和协议从 Brannelly et 2017对等 J22中改编而成.

詹姆斯库克大学批准了应用程序中的动物伦理学 A1875 corroboree以及低压阿尔宾娜的 A1897 和 A2171。
1. 畜牧业和监测
<ol><li>在适宜于该物种的环境中单独饲养动物, 并提供适当的水、喂养和清洁计划。每天检查动物。<ol>
<li>使用严重濒危的Pseudophryne corroboree (用于隧洞化验、4节) 和受威胁的Litoria verreauxii 阿尔宾娜(Caspase 3/7 化验, 5 节), 捐赠自圈养育种设施的成年个人。保持动物在 15-18 °c, 在苔藓和砾石基板上, 每天用反渗透水薄雾他们, 并喂养3次每周与肠道加载蟋蟀。</li>
	</ol>
</li>
	<li>每周收集每个个人的数据。按照步骤2.1 中的说明拭去Bd的个人。使用刻度将每只动物体重接近0.1 克, 并用表盘卡尺测量它们的鼻口以排气 (逸诚) 长度到最近的0.01 毫米。</li>
	<li>接种后 (如第3节所述), 遵守动物伦理准则, 每天检查动物是否有感染的临床症状 (不规则的皮肤脱落、不振、腿红肿、昏睡或延迟矫正反射)。弄死动物, 如果临床症状和发病率存在。<ol>
<li>弄死与过量的 tricaine 甲磺酸 (MS222) (0.1% 瓦特/v MS222 pH 6-7 与碳酸氢钠在老年自来水)。保持动物在 MS222 10 分钟后, 所有运动停止, 他们不显示反应的刺激。</li>
	</ol>
</li>
</ol>2. 测试Bd感染
<ol><li>为Bd擦洗
	<ol>
<li>用一种新的不育人造丝拭子擦拭每只动物 (每只动物一拭子)。使用下面的抽汲方法: 在温特上的五次, 每大腿, 一侧和肢体的五次。这是总共45笔。</li>
		<li>轻轻地旋转拭子在中风期间和之间, 以确保有效地捕获 DNA。将拭子尖端分解成1.5 毫升离心管, 并贮存在-20 °c, 直到萃取。</li>
	</ol>
</li>
	<li>DNA 提取与 qPCR 检测<ol>
<li>通过添加50µL 的 30-40 毫克的0.5 毫米二氧化硅珠, 从拭子中提取基因组 dna。珠打在一个珠子搅拌器细胞内分泌在最大速度为2分钟的样品。下面的珠搅拌器步骤, 离心机的样品在 2000 x g 1 分钟。</li>
		<li>将样品在100摄氏度处孵化10分钟, 并允许冷却。离心样品在 5000 x g 3 分钟, 然后转移上清到单独1.5 毫升微型离心机管子并且存放在-20 °c 直到 qPCR。</li>
		<li>使用定量 PCR (qPCR) 跟随博伊尔et 。200418分析感染负荷。使用以下修改:<ol>
<li>用双去离子水稀释 DNA 萃取样品6:100。添加0.7 µL 的牛血清白蛋白 (BSA), 以帮助防止 PCR 抑制。在 singlicate 中运行每个示例。在每个板块使用一个负 (无模板) 控制和一个积极的控制与一系列稀释标准, 以估计游动孢子 (感染) 负荷。</li>
		</ol>
</li>
	</ol>
</li>
</ol>3. 接种
<ol><li>区域性Bd
	<ol>
<li>加入16克胰蛋白胨, 2 克明胶水解物, 4 克乳糖 (TGHL) 为1升的去离子水, 制备培养液。蒸压釜 (121 °c 40 分钟), 并允许汤冷却。</li>
		<li>接种Bd隔离 (在这种情况下, 从新南威尔士隔离, AbercrombieR booroologensis-2009-LB1, 通道号 11) 在 TGHL 汤和增长 7 d 在23摄氏度, 然后转移汤文化 TGHL 琼脂板块。</li>
		<li>要制作板材, 添加16克胰蛋白胨, 2 克明胶水解物, 4 克乳糖 (TGHL), 和10克细菌琼脂, 以 1 L 的去离子水和高压釜 (121 摄氏度为40分钟)。一旦混合物冷却足够的接触, 但在固化之前, 倒入 TGHL 琼脂到92毫米直径培养板, 所以他们是1/4 到1/3 充分, 在第二类生物安全柜。</li>
		<li>当琼脂完全凝固和冷却后, 从液体汤中接种0.5 毫升的Bd , 并均匀地传播。允许Bd肉汤混合物在板材上干燥约1小时, 然后用塑料石蜡膜密封板。孵育盘琼脂边在23°c 为 5-7 d。</li>
	</ol>
</li>
	<li>游动孢子接种液的洪水板<ol>
<li>检查游动孢子在倒置光显微镜下的运动, 以确保在接种前每天的生存能力。当游动孢子释放很高的时候, 许多孢子在孢子囊外游泳, 这种文化就可以接种了。</li>
		<li>用3毫升的旧自来水或人工池塘水, 将水倒入盘子中, 在室温下孵化10分钟, 使孢子释放到水中。孵化期后, 醒酒游动孢子悬浮液进入新的无菌容器。</li>
		<li>根据制造商的说明使用 haemocytometer 估计游动孢子浓度。一旦已知的游动孢子悬浮液浓度, 稀释悬浮液浓度为 1 x 10 6 孢子每3毫升与老自来水.</li>
	</ol>
</li>
	<li>接种动物<ol>
<li>在各自的50毫升容器中, 通过将3毫升的接种混合物灌注到其在它的温特19上, 接种每种动物。允许过剩的接种收集到接种容器的底部。将每只动物放在单独的接种容器中24小时以确保感染, 在24小时接种后, 将每个个体送回消毒缸。<ol>
<li>使用13% 卷/卷 (v/v) 商用漂白剂解决方案20消毒 terraria, 至少用水冲洗两次, 然后允许干燥不少于24小时。</li>
		</ol>
</li>
		<li>对于Bd-阴性控制, 模拟接种动物使用相同的方法, 在 3.2-3.3, 但洪水bd-免费琼脂板与5毫升的老年自来水, 而不是Bd接种琼脂板。</li>
	</ol>
</li>
</ol>4. 隧洞试验
<ol><li>弄死动物显示壶菌病的临床症状, 如步骤1.3.1 中所述, 以及同样数量的Bd-阴性控制动物。</li>
	<li>解剖皮肤 (背, 腹, 大腿) 标本从每一个动物。修复2小时的皮肤样品在 4% v/v 磷酸盐缓冲甲醛。短而一致的固定时间允许组织完全固定, 但也允许有效和准确的免疫组化染色。然后转移到80% 乙醇, 直到嵌入切片。</li>
	<li>采用标准方法21, 将皮肤嵌入石蜡中进行组织学准备。简言之, 该议定书如下:<ol>
<li>在一系列乙醇中脱水组织, 用二甲苯清除乙醇。在石蜡中嵌入组织, 并将所有三个皮肤样本放置在一个石蜡块中。</li>
	</ol>
</li>
	<li>切片皮肤使用切片以下标准组织学准备21。切片的组织在5µm, 然后贴在亲水性玻璃幻灯片。每张幻灯片的每个皮肤类型放置四个串行 histosections。每个人用系列皮肤切片制作三张幻灯片, 请记住每张幻灯片上贴有四节的每个组织。</li>
	<li>按以下顺序着色幻灯片:<ol>
<li>用苏木精染色第一张幻灯片, 后跟 counterstaining (H & E)。此幻灯片用于可视化Bd孢子囊在皮肤中的位置。</li>
		<li>染色的第二个幻灯片后, 商业上可用的隧洞化验的组织学准备, 并遵循制造商的指示, 其中描述如下。<ol>
<li>首先, deparaffinize coplin 罐中的组织部分, 通过洗涤三二甲苯的变化, 每洗5分钟, 并遵循100% 乙醇的两个变化, 每洗5分钟。遵循一洗95% 乙醇3分钟, 然后70% 乙醇3分钟完成与一洗涤 PBS 为5分钟。</li>
			<li>预处理的组织与新鲜稀释蛋白消化酶 (蛋白酶 K 浓度为20微克/毫升稀释在 PBS), 并直接添加到幻灯片。允许孵育15分钟, 在一个 coplin 罐子的两个变化的 PBS, 每2分钟。</li>
			<li>在室温下, 用 coplin 罐中的3.0% 过氧化氢在 PBS 中轻击多余的液体和淬火, 以2分钟的温度。用 PBS 冲洗两次, 每次洗五分钟。</li>
			<li>轻敲多余的液体, 然后将平衡缓冲器的75µL/5 厘米2直接应用到幻灯片上的组织上。孵化十年代. 在该节周围轻敲多余的液体, 并应用55µL/5 厘米2工作终端 deoxynucleotidyl tranferase (TdT) 酶。在湿润室孵育1小时, 在37摄氏度。</li>
			<li>TdT 孵化后, 将滑块放在 coplin 的工作强度停止/洗涤缓冲液罐中, 搅拌十五年代, 室温孵育10分钟。用三的 PBS 来清洗幻灯片, 每洗1分钟。轻敲多余的液体。</li>
			<li>应用抗辛共轭 (罗丹明) 已被加热到室温的组织, 65 µL/5 厘米2。在室温下孵育30分钟, 避免接触光线。洗涤与四变化 PBS 2 分钟每洗涤。轻敲多余的液体。</li>
			<li>完成通过添加15µL 0.5-1 µg/毫升 DAPI (4 ', 6-diamidino-2 苯基吲哚) 在幻灯片上的幻灯片, 这作为一个反污点。然后盖上盖子滑动和密封用指甲油或橡胶水泥。允许幻灯片在黑暗中干燥, 因为化验是轻敏感的。</li>
		</ol>
</li>
		<li>着色的第三张幻灯片是染色使用的隧洞化验和 DAPI counterstain, 但作为一个积极和消极的控制每个样本。 注意: 在控制幻灯片上的 4 histosections 中, 前2个是正控制复制, 最后2是负控制复制。<ol>
<li>对于阳性对照, 代替步骤 4.5.2.2, 使用 DN 缓冲区 (30mM trizma 基、pH 值7.2、4mM 氯化镁2、0.1mM 滴滴涕) 预先处理组织, 并让室温孵育5分钟。然后将 Dnase I 在 DN 缓冲区中溶解, 以最终集中 0.1ug/mL, 并将其直接应用于幻灯片。室温孵育15分钟。 然后, 用5洗涤 dH2O 清洗幻灯片, 每次清洗3分钟。把多余的液体涂抹掉。然后按照4.5.2.3 节的指示继续进行隧洞化验。</li>
			<li>对于负控制, 不要将终端 deoxynucleotidyl tranferase (TdT) 酶添加到这些样品中 (如4.5.2.4 节所述)。</li>
		</ol>
</li>
	</ol>
</li>
	<li>在化验完成后立即观察。<ol>
<li>使用荧光显微镜, 在200X 的每个皮肤部分随机间隔拍摄照片, 同时使用用于罗丹明染色的过滤器, 它可以揭示凋亡细胞并出现红色, DAPI 揭示所有细胞核并呈蓝色, 从而覆盖可以生成单元格。确保每个样本每皮肤部分至少有100个细胞被拍照。将幻灯片存储在-20 摄氏度的深色显微镜盒中。</li>
		<li>每个图像的计数单元格 (蓝色 DAPI 染色)。确保每个皮肤部分至少有100个细胞计数。要达到100个单元格, 请对图像中的所有单元格进行计数。如果在一个图像中存在少于100个单元格, 则数另一幅图像, 直到每只动物每皮肤部分达到至少100个细胞。</li>
		<li>接下来, 计算同一图像中的隧洞阳性细胞数 (红色罗丹明染色)。指细胞凋亡, 而不是坏死或背景, 是细胞边缘透明的单细胞 (膜不完整, 无裂解)。有时细胞收缩以形成凋亡体。</li>
		<li>定位在 histosections 中计数的区域, 并对 H & E 染色的相应区域进行分析。确认 H & E 染色的部分与bd感染的站点相对应, 这可以确保在对该检测中的凋亡细胞计数时使用bd感染的站点。</li>
	</ol>
</li>
</ol>5. Caspase 3/7 化验
<ol><li>收集每个动物的脚趾提示 ( bd暴露和bd阴性) 每周3次接种后, 每两周通过实验结束, 每个个体最多8个脚趾。<ol>
<li>在第二指骨用火焰灭菌剪刀切开脚趾头, 并放置在1.5 毫升微细, 并立即冻结在-80 摄氏度。</li>
	</ol>
</li>
	<li>从冷冻脚趾样品中提取蛋白质。<ol>
<li>将样品放入100µL 样品缓冲器 (25 毫米 HEPES pH 7, 5 毫米氯化镁2), 并在3.2 毫升螺钉帽微细中使用两个不锈钢珠 (1.5 毫米)。溶解样品由4个周期1极小的珠击在最大速度 (在同一个在步1.2.1 使用的珠子搅拌器) 跟随3分钟在冰。溶解后, 离心机样品在 1.2万 x g 和4°c 为5分钟, 收集上清在化验中使用。</li>
	</ol>
</li>
	<li>使用布拉德福德测定法定量分析每个样品的蛋白质浓度。<ol>
<li>在384井板, 混合10µL 蛋白质提取物与10µL 的布拉德福德试剂, 并孵化2分钟室温。重复执行每个样品, 连同一系列5倍 BSA 稀释标准。阅读吸收率在 595 nm 的吸光度板阅读器。</li>
	</ol>
</li>
	<li>或者, 如果脚趾尖端样品太小 (蛋白质浓度接近最低标准, 从布拉德福德试验中确定的步骤 4.3.1, 或如果被取样的青蛙是低于3克总重量), 通过估计皮肤表面标准化样品区域使用照片, 而不是布拉德福德化验。<ol>
<li>在从样品中提取蛋白质的 caspase 化验之前, 用倒置的光显微镜拍摄每个脚趾的40X 放大倍数。</li>
		<li>使用计算机成像软件分析脚趾的样本, 通过估算脚趾的面积。通过在脚趾夹的边缘上画一条线来完成此项, 并在形状内有图像软件估计区域。然后乘以 pi (3.14) 的面积, 这将近似3维皮肤样品的表面积 (长度 x 横截面积 (pi x 直径) 给出管的外表面面积)。</li>
	</ol>
</li>
	<li>执行 caspase 3/7 化验。<ol>
<li>在384井发光板, 添加10µL 的商业可用 caspase 3/7 试剂和10µL 蛋白质提取物。每三个样本运行一次。</li>
		<li>在十五年代慢慢摇动盘子, 混合试剂. 在室温下将盘子从光中孵化30分钟。使用发光板读取器测量发光。</li>
	</ol>
</li>
</ol>

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作者声明没有竞争的财政利益。

我们探讨了表皮细胞凋亡和细胞死亡作为一种潜在的病理机制的致命疾病壶菌病或机制的抗病能力的Bd 敏感物种。我们使用两种方法评估表皮细胞死亡, 对原位表皮细胞死亡分析的 caspase 法, 以及在整个感染过程中监测表皮细胞死亡的3/7 项试验。我们发现, 细胞死亡和凋亡与感染负荷相关, 细胞死亡在感染部位明显增高。早期感染时, 表皮细胞凋亡减少, 但随着感染组织内病原菌负担的增加, 细胞凋亡增加。
细胞凋亡可以用多种不同的方法进行测试, 每一种都有好处和局限性。用多种方法研究细胞凋亡和细胞死亡是重要的, 以证实其结果。在本研究中, 我们探讨了两种不同的检测, 以观察壶菌病的青蛙表皮细胞死亡和凋亡。首先, 我们试行的方法, 以评估细胞死亡原位。此测试耗时, 并且每个示例都很昂贵, 并且当荧光信号迅速消退时, 必须立即读取幻灯片。尽管存在这些限制, 但此就地实验的结果对于进一步了解宿主病原体相互作用的机制是很重要的。有关本地化的信息可以用这种方法来确定, 在我们的研究中, 细胞凋亡在真菌感染的部位有较高的水平, 其他部位更弥漫。另外它必须注意到, 隧洞化验测量脱氧核糖核酸损伤, 可能由许多不同的细胞死亡机制导致, 包括细胞凋亡, 坏死并且 pyroptosis17。当有细胞凋亡相关的细胞死亡的署名 (如单细胞与膜仍然原封, 看见协议线 4.6.3, 分类依靠研究员的解释。由于细胞死亡的机制不是由隧洞检测来确定的, 因此, 另一种非凋亡细胞死亡通路可能导致了感染动物患病时的隧洞阳性细胞增多。每种化验方法的不同之处可能解释两个化验试行的模式差异。
caspase 3/7 测定法可用于量化感染时间的影响。然而, 由于青蛙脚趾样本是小的, 有有限的样本进行分析和标准化。样本标准化的常用方法是确定每个提取物的总蛋白质浓度。由于布拉德福德蛋白质定量分析法的使用, 对小提取物的研究不是一种有效的方法。此外, 我们发现, 在青蛙皮肤内的色素排除了纳米紫外-可见光谱分析。小样本尺寸也防止了干燥重量的标准化。我们建议用摄影方法估算脚趾的皮肤表面积, 因为脚趾头的样本 (这些青蛙的体型小于3克) 太小, 不能进行蛋白质浓度和 caspase 浓度的测定。我们发现, 拍摄脚趾和使用皮肤表面积估计是一样有效的传统蛋白质浓度分析。
在这项研究中, 我们使用了两种标准的方法来量化细胞死亡和细胞凋亡, 但适应了两栖动物和小组织样本的方法。对于隧洞试验, 我们对动物进行了安乐死, 以确保有足够的组织可供化验, 并允许比较不同的皮肤部位。这种方法可以适应较小的组织样本, 如脚趾织带活检, 这将不需要动物的安乐死。根据动物的大小, 活检可以让时间序列测试类似于我们的方法为 caspase 3/7 化验。
在今后的研究中, 我们建议使用额外的检测方法来探索细胞死亡和凋亡在壶菌病感染过程中, 因为还有很多要学习的因果机制的壶菌病病机。这些结果表明, 凋亡和表皮细胞死亡可能是重要的发病机制的Bd;然而, 为了确定凋亡对疾病结局的影响, 特别是在不屈从于感染的宿主中, 需要进行更多的研究.

两栖动物目前正在经历一个最大的损失全球生物多样性的任何脊椎动物分类1。这些下降的主要原因是致命的皮肤病壶菌病, 由真菌病原体 Batrachochytrium dendrobatidis, Bd 2 引起.病原体表面上感染表皮, 这可能导致皮肤功能的破坏, 导致严重的电解质丧失, 心跳骤停, 死亡3。目前正在研究针对Bd的各种潜在宿主免疫机制, 例如抗菌肽4、5、皮肤细菌菌群6、免疫细胞受体7、8、和淋巴细胞活动9,10。然而, 很少有研究探讨表皮细胞凋亡和细胞死亡是否是对抗这种致命病原体的免疫机制。
细胞死亡, 无论是通过凋亡 (程序性细胞死亡) 或坏死 (unprogrammed 死亡), 在表皮可能是病理学的Bd感染。先前的研究表明, Bd感染可能诱导细胞凋亡, 因为当皮肤外植体暴露在游动孢子上清液体内 11 中时, 细胞内结的破坏就会被观察到。此外, 在Bd感染的青蛙的变性表皮变化观察使用电子显微镜12,13。Transcriptomic 分析表明, 凋亡通路是上调在感染皮肤14, 两栖脾发生凋亡时, 他们暴露在Bd上清液在体外15。尽管越来越多的证据表明, Bd可以诱导细胞凋亡和宿主细胞死亡体外,在体内研究, 探索或量化凋亡机制, 通过感染的进展缺乏。此外, 不知道宿主是否使用凋亡作为防御性免疫策略来对抗Bd感染, 或者如果凋亡是疾病的病理学。
在本研究中, 我们的目的是通过两种方法来检测感染动物的表皮细胞死亡和细胞凋亡: caspase 3/7 蛋白测定, 以及末端转移酶介导的 dUTP 尼克末端标签 (隧洞) 原位测定。由于每种检测都检测到细胞死亡的不同方面16, 这些方法一起提供了对细胞死亡所涉及的机制的充分理解, 并确保准确测量效果。caspase 3/7 的分析量化了效应半胱氨酸蛋白酶3和7的活动, 这使得定量的内在和外在的凋亡途径。与此相反, 该方法检测 DNA 碎片, 这是由细胞死亡机制, 包括凋亡, 坏死和 pyroptosis17造成的。我们使用三种不同的皮肤部位: 背部、温特和Pseudophryne corroboree的大腿, 利用该方法对临床感染和未感染动物表皮内的细胞死亡位置进行调查。该方法确定细胞死亡的解剖部位, 并鉴别其在特定表皮层内的位置。然后, 我们使用 caspase 3/7 检测在Litoria verreauxii 阿尔宾娜中对整个8周的感染进行时间序列的细胞凋亡量化。我们从相同的动物两周的脚趾提示样本, 并能够关联病原体感染负荷与 caspase 3/7 活动。

<table><tbody><tr><td>POLARstar Omega</td><td>BMG Labtech</td><td></td><td>Luminescent plate reader</td></tr><tr><td>384 well flat clear bottom plate</td><td>Corning</td><td>3707</td><td></td></tr><tr><td>384 well low flange white flat bottom plate</td><td>Corning</td><td>3570</td><td></td></tr><tr><td>Agar Bacteriological (Oxoid)</td><td>Fisher</td><td>OXLP0011B</td><td></td></tr><tr><td>Formal-Fixx 10% Neutral Buffered Formalin</td><td>Fisher</td><td>6764254</td><td></td></tr><tr><td>Lactose Broth (Oxoid)</td><td>Fisher</td><td>OXCM0137B</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Fisher</td><td>BP328-500</td><td></td></tr><tr><td>Tricane-S (MS-222)</td><td>Fisher</td><td>NC0872873</td><td></td></tr><tr><td>Tryptone</td><td>Fisher</td><td>BP1421-500</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Invitrogen</td><td>15561020</td><td></td></tr><tr><td>Sterile rayon swab</td><td>Medical Wire &amp;amp; Equipment</td><td>MW-113</td><td></td></tr><tr><td>ApopTag Red In Situ Apoptosis Detection Kit</td><td>Merck Millipore</td><td>S7165</td><td></td></tr><tr><td>Coomassie Bradford reagent</td><td>Pierce</td><td>23200</td><td></td></tr><tr><td>Caspase Glo&amp;nbsp; 3/7</td><td>Promega</td><td>G8090</td><td></td></tr><tr><td>HEPES buffer</td><td>Sigma Aldrich</td><td>H0887-20ML</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>1374248-1G</td><td></td></tr><tr><td>Gelatin hydrolysate Enzymatic</td><td>Sigma-Aldrich</td><td>G0262</td><td></td></tr><tr><td>PBS (Phosphate Buffered Saline), pH 7.2 (1X)</td><td>Thermo/Life</td><td>20-012-043</td><td></td></tr><tr><td>Prepman</td><td>Thermo/Life</td><td>4318930</td><td></td></tr><tr><td>TaqMan Fast Advanced Master Mix</td><td>ThermoFisher</td><td>4444556</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM996</td><td></td></tr><tr><td>Clorox bleach</td><td>Clorox</td><td></td><td></td></tr><tr><td>Ethanol, 200 Proof, Molecular Grade</td><td>Fisher</td><td>BP2818500</td><td></td></tr><tr><td>ZEISS Axio Scan florescent miscroscope</td><td>Carl Zeiss</td><td></td><td>Florescent microscope</td></tr><tr><td>3.2mm stainless steel beads</td><td>BioSpec</td><td>11079132SS</td><td></td></tr><tr><td>Primer ITSI-3 Chytr (5&amp;prime;-CCTTGAT&lt;br /&gt;ATAATACAGTGTGCCATATGTC-3&amp;prime;)</td><td>Taqman</td><td></td><td>Individual design for primers and probe</td></tr><tr><td>Primer 5.8S Chytr (5&amp;prime;-TCGGTT&lt;br /&gt;CTCTAGGCAACAGTTT-3&amp;prime;)</td><td>Taqman</td><td></td><td>Individual design for primers and probe</td></tr><tr><td>Minor groove binder probe Chytr MGB2(5&amp;prime;-CGAGTCGAAC-3&amp;prime;)</td><td>Taqman</td><td></td><td>Individual design for primers and probe</td></tr><tr><td>Rotor-Gene qPCR Instruments</td><td>Qiagen</td><td></td><td>qPCR machine</td></tr><tr><td>Microcentrifuge tubes 1.5ml</td><td>Fisher</td><td>02-681-372</td><td></td></tr><tr><td>Cell culture petri plates</td><td>Nunc</td><td>263991</td><td></td></tr><tr><td>Mini-beadBeater Zircornia-Silicate Beads, 0.5mm</td><td>BioSpec</td><td>11079105Z</td><td></td></tr></tbody></table>

using terminal transferase-mediated dutp nick end-labelling (tunel) and caspase 3/7 assays to measure epidermal cell death in frogs with chytridiomycosis

两栖动物在全球范围内的生物多样性遭受巨大损失, 其中一个主要原因是传染病壶菌病。这种疾病是由真菌病原体Batrachochytrium dendrobatidis (Bd) 引起的, 它感染和扰乱了青蛙表皮;然而, 病理变化没有明确的特征。细胞凋亡 (程序性细胞死亡) 可以被病原体用来损伤宿主组织, 但也可以成为病原体去除抗病的宿主机制。在这项研究中, 我们用两种不同的化验方法来量化感染和未感染动物的表皮细胞死亡: 末端转移酶介导的 dUTP 尼克末端标签 (caspase 3/7)。采用腹壁、背侧和大腿皮肤组织, 观察临床感染动物的表皮细胞中的细胞死亡, 并用荧光显微术比较未感染动物的细胞死亡. 为了确定表皮细胞凋亡水平在感染过程中的变化情况, 我们在8周的时间里去掉脚趾头的样本, 并使用提取的蛋白质 caspase 3/7 化验来量化样品中的活动。然后, 我们将 caspase 3/7 活动与感染负荷相关联。该方法对细胞死亡的局部化有很大的作用就地, 但成本高昂, 且每次取样时间密集。caspase 3/7 法对大样本量和时间过程实验有很高的效率。然而, 由于青蛙脚趾尖端活检是小的, 有有限的提取物可用于样本标准化通过蛋白质量化方法, 如布拉德福德化验。因此, 我们建议通过对脚趾活检的摄影分析来估计皮肤表面积, 以避免在样品标准化过程中消耗提取物。

隧洞试验
感染动物中的细胞比未感染的对照动物多。在感染和控制的动物中, 隧洞阳性细胞的原位位置不同。在控制动物中, 在皮肤和表皮层的低水平上, 有一个均匀分布的隧洞阳性细胞 (参见图 1A), 但在受感染的动物中, 在表皮中的隧洞阳性细胞更频繁 (图1B). 感染部位 (如观察到的 H & E 染色的幻灯片) 被观察为丛生Bd孢子囊分散通过大腿和腹侧皮肤部分没有在背部部分。虽然在感染细胞的部位 (图 1C) 中, 隧洞阳性细胞更集中于和直接相邻, 但在表皮和表皮层也有更广泛的阳性细胞, 比Bd更深。驻留.在Bd受感染的动物中, 所有三种皮肤类型都有更多的隧洞阳性细胞, 但在大腿和温特皮肤 (12.01 倍以上 (95% ci: 4.92-26.30) 的大腿皮肤和22.31 倍以上 (95% ci 5.25-94.82) 中, 有一个特别高的水平 温特皮肤) (图 2)。
Caspase 3/7 化验
在感染过程中, caspase 活动有差异。在Bd受感染的动物中, 感染强度与 caspase 3/7 活动 (图 3) 呈正相关。感染和未感染的动物也有不同的时间, 接种后。Caspase 3/7 活动在接种后的头几周内减少 (在受感染的动物中减少48.36% 的活动), 然后在Bd受感染的动物中增加到7周 (图 4) 的末尾, 但在未感染的情况下保持不变。个人。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57345/57345fig1.jpg"> 图 1: 终端转移酶介导的 dUTP 尼克末端标签 (隧洞) 原位检测感染和未感染的动物.A) Bd-控制Pseudophryne corroboree、和 B) Bd+ 大腿皮肤部分 ( P. corroboree的大腿皮肤部分)原位隧洞检测染色。蓝色是 DAPI 染色表示细胞核的细胞, 红色是罗丹明染色, 这表明 DNA 碎片的典型原因是凋亡。黄色箭头指示Bd群集的位置, 在面板 c. c 中看到) P. corroboree部分的大腿皮肤染色的 H & E。H & E 部分是串行到 B 组。在皮肤表面附近有一组空的Bd孢子囊 (箭头) 和一些暗不成熟的孢子囊. 对于所有三面板表皮是在照片的顶端。比较小组 B 和 C 显示, 罗丹明染色的表皮细胞集中在Bd群的周围和下方, 皮肤损伤可见, 如基底表皮细胞之间的微囊形成。400X 放大倍数和缩放条表示0.03 毫米. 在 Brannelly et . 2017对等 J22中, 适合于图 2 。<a href="/files/ftp_upload/57345/57345fig1large.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/57345/57345fig2.jpg"> 图 2: 每种皮肤类型的隧洞阳性 (隧洞 +) 细胞比例.在P. corroboree中, 每种皮肤的隧洞阳性凋亡细胞的比例, 与受感染的动物, 表明动物屈服于疾病 (n = 9) 和未感染的控制动物 (n = 10).误差线表示95% 置信区间的比例和 * 表明, 在隧洞 + 细胞比例显著增加。在背皮中, 受感染的动物有 14.38 (95% CI 3.32-62.24) 倍于对照动物的细胞 (赔率比: Z = 3.57, p < 0.01)。在大腿皮肤, 被传染的动物有 12.01 (95% 词: 4.92-26.30;赔率比率: Z = 5.46, p < 0.01) 倍的比控制动物更有细胞的阳性单元 (皮尔逊的 Chi 的平方: χ21 = 44.30, p < 0.01)。在温特皮肤, 受感染的动物有 22.31 (95% CI 5.25-94.82) 倍的细胞比对照动物 (赔率比率: Z = 4.21, p < 0.01) 时间更多。从图 3在 Brannelly et . 2017对等 J22中改编。<a href="/files/ftp_upload/57345/57345fig2large.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/57345/57345fig3.jpg"> 图 3: 在实验过程中, 感染强度、日志10(游动孢子等价) 和 caspase 3/7、日志 10 (caspase) 接种Litoria verreauxii阿尔宾娜之间的相关性。感染强度与 caspase 活性的相关性为 0.463, 趋势线有 y = (0.229) x + 0.939 的方程。自 Brannelly et al中的图 4改编而成。2017对等 J22.<a href="/files/ftp_upload/57345/57345fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57345/57345fig4.jpg"> 图 4: 每组Litoria verreauxii 阿尔宾娜: Bd受感染的动物 (n = 4) 和未感染的控制 (n = 8), Caspase 3/7 活动到7周.Caspase 活动被定义为按样品的蛋白质浓度控制的发光读数, 然后对数基10变换。每个组每周的 caspase 活动 (日志10转换)。错误条表示标准错误。* 显示受感染动物与该星期未感染的对照组不同。(线性混合效应模型: 周, F4 = 11.974, p < 0.01; 周 * 状态, F8 = 2.139, p = 0.037;3周方差分析, F218 = 5.512, p = 0.014), 从图 5在 Brannelly等. 2017对等 J22中改编.<a href="/files/ftp_upload/57345/57345fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis at JoV

Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

我们用两种方法定量测定青蛙壶菌病的表皮细胞死亡。首先, 我们使用终端转移酶介导的 dUTP 尼克末端标签 (隧洞)的原位组织学, 以确定临床感染和未感染动物之间的差异。其次, 用 caspase 3/7 蛋白分析方法对感染细胞凋亡进行时间序列分析。

用末端转移酶介导的 dUTP 尼克末端标记 (Caspase 3/7) 测定青蛙壶菌病的表皮细胞死亡

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is ...

using-terminal-transferase-mediated-dutp-nick-end-labelling-tunel

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

10.4K 次观看.  James Cook University. 我们用两种方法定量测定青蛙壶菌病的表皮细胞死亡。首先, 我们使用终端转移酶介导的 dUTP 尼克末端标签 (隧洞)的原位组织学, 以确定临床感染和未感染动物之间的差异。其次, 用 caspase 3/7 蛋白分析方法对感染细胞凋亡进行时间序列分析。

视频: Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

利用分子荧光互补的方法照亮 Caspase 活化的途径

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

科研

生物化学

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

监测劈开caspase - 3活性和钾诱导膜去极化使用活细胞成像和Cleaveable荧光染料基板的永生的少突胶质细胞凋亡

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

神经科学

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

使用荧光测定法或流式细胞术测量半胱天冬酶活性

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

用 CaspaseTracker 生物传感器检测 Anastasis体内

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

医学

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

生物学

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.
This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including&#160;apoptosis,&#160;pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and&#160;cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and&#160;leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases,&#160;from other cell death pathways.
Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

评估半胱天冬酶活化以评估先天免疫细胞死亡

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is ...

免疫与感染

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

体内生物传感器跟踪非凋亡胱天蛋白酶的活动在果蝇

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...

The TUNEL Assay

One of the hallmarks of apoptosis is the nuclear DNA fragmentation by nucleases. These enzymes are activated by caspases, the family of proteins that execute the cell death program. TUNEL assay is a method that takes advantage of this feature to detect apoptotic cells. In this assay, an enzyme called terminal deoxynucleotidyl transferase catalyzes the addition of dUTP nucleotides to the free 3&#8217; ends of fragmented DNA. By using dUTPs that are labeled with chemical tags that can produce fluorescence or color, apoptotic cells can be specifically identified. JoVE&#8217;s video on the TUNEL assay begins by discussing how this technique can be used to detect apoptotic cells. We then go through a general protocol for performing TUNEL assays on tissue sections and visualizing the results using fluorescence microscopy. Finally, several applications of the assay to current research will be covered.

应用 TUNEL 法检测

One of the hallmarks of apoptosis is the nuclear DNA fragmentation by nucleases. These enzymes are activated by caspases, the family of proteins that ...

教育

JoVE Science Education

高级生物学

细胞生物学

Terminal Transferase-Mediated dUTP Nick End-Labeling&#8212;TUNEL Assay: An In Situ Method to Detect DNA Fragmentation in Apoptotic Cells

Source: Brannelly, L. A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57345">Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis.</a> J. Vis. Exp. (2018).
In this video, the TUNEL assay is performed to detect the presence of apoptotic cells in a sample. The study involves using a special DNA polymerase - terminal deoxynucleotidyl transferase - that catalyzes the addition of labeled dUTPs to the 3&#39; terminus of fragmented DNA, which can be observed and identified under a fluorescence microscope.

末端转移酶介导的 dUTP 缺口末端标记 - TUNEL 检测:一种检测凋亡细胞中 DNA 片段化的原位方法

Source: Brannelly, L. A., et al. Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell ...