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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F., Berger, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chytridiomycosis+on+hematopoietic+tissue+in+the+spleen,+kidney+and+bone+marrow+in+three+diverse+amphibian+species.">Effects of chytridiomycosis on hematopoietic tissue in the spleen, kidney and bone marrow in three diverse amphibian species.</a> Pathogens and Disease. 74 (7), ftw069 (2016).</li><li>Brutyn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Batrachochytrium+dendrobatidis+zoospore+secretions+rapidly+disturb+intercellular+junctions+in+frog+skin.">Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin.</a> Fungal Genetics and Biology. 49 (10), 830-837 (2012).</li><li>Berger, L., Hyatt, A. D., Speare, R., Longcore, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+stages+of+the+amphibian+chytrid+Batrachochytrium+dendrobatidis.">Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis.</a> Diseases of Aquatic Organisms. 68 (1), 51-63 (2005).</li><li>Pasmans, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chytridiomycosis+related+mortality+in+a+midwife+toad+(Alytes+obstetricans)+in+Belgium.">Chytridiomycosis related mortality in a midwife toad (Alytes obstetricans) in Belgium.</a> Vlaams Diergeneeskundig Tijdschrift. 79 (6), 460-462 (2010).</li><li>Ellison, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+than+skin+deep:+Functional+genomic+basis+for+resistance+to+amphibian+chytridiomycosis.">More than skin deep: Functional genomic basis for resistance to amphibian chytridiomycosis.</a> Genome Biology and Evolution. 7 (1), 286-298 (2014).</li><li>Fites, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+invasive+chytrid+fungus+of+amphibians+paralyzes+lymphocyte+responses.">The invasive chytrid fungus of amphibians paralyzes lymphocyte responses.</a> Science. 342 (6156), 366-369 (2013).</li><li>Galluzzi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+cell+death+in+higher+eukaryotes.">Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.</a> Cell Death and Differentiation. 16 (8), 1093-1107 (2009).</li><li>Kelly, K. J., Sandoval, R. M., Dunn, K. W., Molitoris, B. A., Dagher, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+determine+specificity+and+sensitivity+of+the+TUNEL+reaction+in+the+quantitation+of+apoptosis.">A novel method to determine specificity and sensitivity of the TUNEL reaction in the quantitation of apoptosis.</a> American Journal of Cell Physiology. 284 (5), C1309-C1318 (2003).</li><li>Boyle, D. G., Boyle, D. B., Olsen, V., Morgan, J. A. T., Hyatt, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantitative+detection+of+chytridiomycosis+(Batrachochytrium+dendrobatidis)+in+amphibian+samples+using+real-time+Taqman+PCR+assay.">Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay.</a> Diseases of Aquatic Organisms. 60 (2), 141-148 (2004).</li><li>Brannelly, L. A., Webb, R., Skerratt, L. 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A., Roberts, A. A., Skerratt, L. F., Berger, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+cell+death+in+frogs+with+chytridiomycosis.">Epidermal cell death in frogs with chytridiomycosis.</a> PeerJ. 5, e2925 (2017).</li></ol>

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...

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 &#38; 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&#160; 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&#8242;-CCTTGATATAATACAGTGTGCCATATGTC-3&#8242;)</td>
			<td>Taqman</td>
			<td></td>
			<td>Individual design for primers and probe</td>
		</tr>
		<tr>
			<td>Primer 5.8S Chytr (5&#8242;-TCGGTTCTCTAGGCAACAGTTT-3&#8242;)</td>
			<td>Taqman</td>
			<td></td>
			<td>Individual design for primers and probe</td>
		</tr>
		<tr>
			<td>Minor groove binder probe Chytr MGB2(5&#8242;-CGAGTCGAAC-3&#8242;)</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...

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 ...

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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

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

竞争的原点实验研究肠嗜T细胞迁移

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

科研

免疫与感染

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

肠道细菌侵入肠上皮细胞<em&gt;体外</em&gt;使用垂直扩散室模型，显着增强

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. Products are routinely stored at 22 &#176;C on an agitating shaker, a condition that can promote bacterial growth. Although the total number of bacteria believed to be introduced into a platelet product is extremely low, these bacteria can multiply to a very high titer prior to transfusion, potentially resulting in serious adverse events. The aim of this study was to evaluate a riboflavin based pathogen reduction process against a panel of bacteria that have been identified as common contaminants of platelet products. This panel included the following organisms: S. epidermidis, S. aureus, S. mitis, S. pyogenes, S. marcescens, Y. enterocolitica, B. neotomae, B. cereus, E. coli, P. aeruginosa and K. pneumoniae. Each platelet unit was inoculated with a high bacterial load and samples were removed both before and after treatment. A colony forming assay, using an end point dilution scheme, was used to determine the pre-treatment and post-treatment bacterial titers. Log reduction was calculated by subtracting the post-treatment titer from the pre-treatment titer. The following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli &#8805;5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%). The results from this study suggest the process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

治疗核黄素血小板制品和紫外线光:对有效性高滴度细菌污染

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

利用X射线晶体学，生物物理学，和功能分析，以确定管理机制T细胞癌抗原的受体识别

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is based on the injection of non-pathogenic particles into insects, as the particles will be detected by the immune system and will induce the production of immune effectors. We focus here on the stimulation of the melanization response in the mosquito Anopheles gambiae. The melanization response results in the encapsulation of foreign particles and parasites with a dark layer of melanin. To stimulate this response, mosquitoes are inoculated with beads in the thoracic cavity using microcapillary glass tubes. Then, after 24 hr, the mosquitoes are dissected to retrieve the beads. The degree of melanization of the bead is measured using image analysis software. Beads do not have the pathogenic effects of parasites, or their capacity to evade or suppress the immune response. These injections are a way to measure immune efficacy and the impact of immune stimulations on other life history traits, such as fecundity or longevity. It is not exactly the same as directly studying host-parasite interactions, but it is an interesting tool to study immunity and its evolutionary ecology.

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

接种<em&gt;冈比亚按蚊</em&gt;珠蚊子诱发和测量黑化免疫反应

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...