KP 和写和修订的手稿。KP 进行了实验和分析。这项工作得到了国家卫生研究院 (NIH) 职业过渡奖 (K22AI112816) 的支持。

所有实验程序都是在加利福尼亚大学欧文分校进行的。
1. 缓冲器和解决方案
<ol><li>在1升玻璃瓶中加入25克的溶源性汤 (磅), 在1升的双蒸馏水 (ddH2O) 中添加一个磅米勒混合物。对于培养皿, 添加额外的20克琼脂。灭菌釜。将25毫升的熔融琼脂培养基倒入10厘米直径的培养皿中, 并允许室温固化。将液体介质存放在室温和琼脂板上, 温度为4摄氏度。</li>
	<li>在1升玻璃瓶中混合1克葡萄糖, 制备葡萄糖酵母蛋白胨 (别扭) 板, 2 克的蛋白胨, 0.25 克酵母提取物, 4.2 克的2PO4, 5.1 克的 Na2HPO47H2O, 25 克的琼脂在1升 ddH2o. 高压釜消毒。将25毫升的熔融琼脂倒入10厘米直径的培养皿中, 并允许室温固化。存储在4摄氏度。</li>
	<li>用10 克蛋白胨、7克酵母提取物、15克 d-葡萄糖、0.12 克钠2HPO47H2O、1.4 g 的µg 2 4、40的维生素 B12的1升玻璃瓶制备蛋白胨 S (PS) 培养基, 80 的叶酸在800毫升的 ddH2o) 校准电子 ph 表使用 ph 值4和 ph 7 标准, 如果 ph 计没有在一天内使用。<ol>
<li>使用 ph 计测量 PS 介质的 ph 值。添加5米 KOH 或5米 H3PO4调整 pH 值为6.5。将最终音量调整为1升, 使用 ddH2o 过滤器消毒0.22 µm 真空过滤器, 并存储在4摄氏度。 注意: 通过佩戴防护设备, 包括手套、防护服、护眼和脸部防护, 谨慎地进行 pH 调整。</li>
	</ol>
</li>
	<li>在1升玻璃瓶中, 通过添加3.4 克2PO4、13.4 克 Na2HPO4 7H2o 和 ddH2o 至800毫升的总容积, 准备10x 开发缓冲器素数 (DB) 缓冲器。如果 ph 计没有在一天内使用, 请使用 ph 值4和 ph 值7标准校准电子 ph 值表。<ol>
<li>使用电子 pH 计测量缓冲器的 ph 值。根据需要轻轻添加5米或5米3PO4 , 将 pH 值调整为6.5。将最终音量调整为1升, 使用 ddH2O, 热处理消毒。在室温下贮存。 注意: 通过佩戴防护设备, 包括手套、防护服、护眼和脸部防护, 谨慎地进行 pH 调整。</li>
	</ol>
</li>
	<li>使1x 开发缓冲区 (DB) 混合100毫升 10x DB 的缓冲器与1毫升灭菌2米氯化镁2, 和1毫升灭菌 1 m CaCl2。使用蒸压灭菌 ddH2o 在室温下, 将最终音量调整为1升玻璃瓶中的1升。</li>
	<li>准备 PS: 1 升玻璃瓶中的 DB 介质, 混合100毫升灭菌 PS 培养基, 100 毫升灭菌的 10x DB 缓冲液, 并灭菌 ddH2O 到总容积 1 l 补充与1毫升灭菌 2 m 氯化镁2和1毫升灭菌 1 m CaCl2. 贮存在4摄氏度。</li>
	<li>在制造商提供的塑料容器中加入50µL 的亚砜, 在 calcein 的50µg 中, 准备 calcein 的库存解决方案。存储在-20 摄氏度。</li>
</ol>2. 阿米巴的生长和维持
<ol><li>生长大肠杆菌菌株17作为阿米巴的食物来源在最初的成长期。将大肠杆菌从贮存于-80 摄氏度的冰冻储存到 LB 琼脂板上, 在一夜之间孵化37摄氏度以获得单一菌落。</li>
	<li>将大肠杆菌的单个菌落接种成2毫升的 LB 培养基, 在滚筒滚筒或37摄氏度的振动筛中孵化一夜。</li>
	<li>把隔夜大肠杆菌培养到室温。使用木棍, 接种阿米巴从冻结的库存保持在-80 °c 到750µL 的隔夜文化。 注: 使用D. 柄菌株 AX3, 无菌生长能力为18。这一步不需要无菌增长, 但随后的步骤将有他的要求。</li>
	<li>立即将阿米巴大肠杆菌混合物的700µL 添加到葡萄糖酵母蛋白胨 (别扭) 板上。将文化均匀地放在盘子的表面上, 使其在需要的时候向后倾斜和侧向一侧。避免使用摊铺机。</li>
	<li>将别扭板与琼脂正面朝上, 保持高湿度。使用两个一次性塑料容器 (183 厘米 x 183 厘米 x 91 厘米) 堆叠在彼此的顶部。用大约25毫升的水填充底部容器, 并将顶部容器与0.5 厘米直径的孔一起钻入容器的地板上, 允许湿气从蓄水池移动到顶容器。</li>
	<li>孵化的别扭板和盒在22°c 4-5 天, 直到变形虫孢子被观察到生长在板块表面附近 (图 2)。使用冷藏孵化器孵化板块, 而不是在室温下孵化。 注: 在孢子生长后, 它们在盘子中的存活时间在4摄氏度时保持不变。将印有琼脂面的盘子朝上。经过 3-4 天的孵化, 可以观察到细菌草坪内的清除区, 这表明孢子虫的发病。<ol>
<li>观察 4-5 天的孵化后, 在板表面上方的小阿米巴孢子 (图 2)。 注意: 不要长超过一周的阿米巴, 因为孢子的质量在这个时间段内会减少。</li>
	</ol>
</li>
	<li>收集阿米巴孢子, 以准备无菌生长的阿米巴通过扫一个不育的1毫升吸管尖端平行的板表面。收集10厘米培养皿的一半孢子。 注意: 避免接触到琼脂盘表面的尖端, 因为这项运动将收集大量大肠杆菌。</li>
	<li>并用重悬将尖端浸入1毫升的 PS 培养基中, 轻轻吹打上下, 将阿米巴放在吸管尖端。</li>
	<li>将接种后的混合物转化为含有25毫升 PS 培养基的250毫升烧瓶, 辅以0.25x 浓度的抗生素防霉溶液。 注: 将抗生素防霉溶液贮存为250µL 整除数-20 摄氏度。避免重复循环的解冻和冻结这个解决方案, 因为这可能会降低其功效。</li>
	<li>生长阿米巴 axenically 在22°c 在旋转振动筛在100转每分钟。对于步骤5中的毒力检测, 将细胞生长成光学密度, 测量 600 nm (OD600) 0.2 到0.5。 注: 这一步骤需要 3-4 天的增长从接种时间 (步骤 2.7)。如果细胞生长到更高的密度, 将培养基稀释到600的0.05 或更低, 然后长到600到0.2 到0.5 的 od。</li>
</ol>3. 铜绿假单胞菌的生长
<ol><li>从 LB 盘中挑选一只单胞菌, 将其接种成2毫升 PS: DB 培养, 并在37摄氏度一夜之间生长到饱和度。 注意: 不要将抗生素防霉溶液添加到 PS: DB 介质中。</li>
	<li>稀释隔夜文化1:100 或 1:1, 000 成6厘米直径培养皿使用 PS: DB。如果浮游细胞的毒力仅被测定, 那么就用传统的试管来培养它们。摇动的文化在一个台式旋转设置在 100 rpm 在37摄氏度。</li>
	<li>收获文化在特定的时间点, 以确保重现性。在评估毒力前30分钟到1小时, 准备一个琼脂垫, 如第4节所述。 注意: 铜绿假单胞菌的毒力是由表面大约8小时的生长引起的。</li>
	<li>要隔离表面附着的细胞, 从培养皿中取出液体培养基, 立即用1毫升的 DB 缓冲器清洗, 以去除浮游细胞, 然后立即进入步骤5.1。 注意: 不要洗涤长于三十年代, 因为这将扰乱和分离表面附着细胞, 可能会影响毒力测量。</li>
	<li>要隔离浮游细胞, 将10µL 的培养物从培养皿转移到干净的培养皿中, 然后立即进行步骤5.1。</li>
</ol>4. 显微术. 琼脂垫的制备
<ol><li>在阿米巴准备与铜绿假单胞菌混合之前, 准备琼脂垫约30分钟至1小时。</li>
	<li>微波50毫升 1% (瓦特/v) 琼脂在 DB 缓冲器在250毫升瓶。每二十年代混合, 直到琼脂丛消失。</li>
	<li>将熔融琼脂放在预热的55°c 水浴中冷却15分钟。</li>
	<li>添加15µL 的 calcein 的股票溶液到15毫升的熔融琼脂在锥形管。混合通过轻轻地反转管 4-5 次, 并立即进行下一步。 注: 在聚丙烯15毫升离心管中执行此步骤。不要使用厚玻璃容器, 因为这会使琼脂凝固得太快。</li>
	<li>将熔融琼脂混合物倒入12厘米 x 10 厘米的玻璃板上, 使琼脂在室温下凝固10分钟。通过将玻璃板放在印刷网格上, 并用金属尺沿网格切割, 将凝固琼脂垫切成较小的1.5 厘米 x 1.5 厘米的部分。</li>
	<li>保持凝胶在潮湿的盒子里水合, 直到它可以使用。</li>
</ol>5. 显微术. 细菌-变形虫样的制备. 成像
<ol><li>为了检测表面附着细菌细胞的毒力, 从步骤3.4 中添加10µL 的变形细胞, 从2.10 步到表面附着细菌。 注: 本步骤描述了阿米巴与细菌的混合。</li>
	<li>为了测定浮游细胞的毒力, 从步骤2.10 中将10µL 的变形细胞与10µL 的浮游细菌混合在步骤3.5 中。 注: 在与细菌混合之前, 不要清洗 axenically 生长的变形虫细胞 (在600到0.2 到0.5 的 OD)。由于使用了抗生素防霉溶液, 在变形虫的培养中不会发现大肠杆菌的生长。</li>
	<li>立即把1.5 厘米 x 1.5 厘米 calcein 琼脂垫从步骤4.5 上的细菌-变形虫混合物上的培养皿表面上。 注: 使用快速平滑运动将琼脂垫放在混合物的顶部。不要缓慢地降低垫在混合物, 因为这项运动倾向于推动细胞向外围和远离垫的底部。这一步骤将确保细菌和阿米巴均匀混合, 固定化, 并且两种细胞类型被限制在同一平面上。</li>
	<li>通过轻轻吹打在琼脂垫周围残留的液体, 去除多余的液体。允许培养皿在室温下干燥20分钟。</li>
	<li>盖上带盖子的培养皿, 在室温下孵化固定化的变形虫-细菌混合物, 再加40分钟. 继续步骤6.1。 注意: 在相同的时间内孵化所有样本是很重要的, 因为背景荧光随着时间的推移而增加。推荐1小时的孵化时间。孵化超过1小时的重复性较差, 因为阿米巴细胞可能开始爆裂。</li>
</ol>6. 显微图像采集
<ol><li>图像阿米巴使用荧光显微镜, 能够获得 brightfield 和绿色荧光图像。对于绿色荧光, 分别使用 474/27 nm 和 525/45 nm 的绿色荧光蛋白 (GFP) 过滤器进行激发和发射。使用 10X (≥0.3 数字光圈) 的目标来图像的阿米巴。 注: calcein 在琼脂中会导致阿米巴在绿色荧光通道中的荧光。健康的阿米巴否则不是荧光的。</li>
	<li>调整相衬的采集时间, GFP 通道限制毒性, 优化图像强度的动态范围。如有必要, 替换差动干涉对比 (DIC) 相衬。 注: 如果可能, 使用 100-200 ms 曝光的相位对比和 GFP 荧光。在整个实验中保持曝光率和仪器设置不变。这对于计算主机杀死索引非常重要。</li>
	<li>获取至少100个变形虫细胞的图像, 以获得计算宿主死亡指数的良好统计数据。</li>
</ol>7. 图像分析和主机杀伤指数
<ol><li>使用 ImageJ 进行图像分析。</li>
	<li>在 ImageJ 中打开相位对比度图像文件, 点击文件 |打开并选择图像。点击图像调整阈值|分析 |门槛。调整下下限和上阈值, 只选择强度峰值 (图 3A)。</li>
	<li>单击 "处理" | "转换为二进制图像" 二进制 |做二进制。通过选择过程填充孔|二进制 |填充孔。 注意: 使用图 1中的图像作为源, 步骤 7.1-7.2 将产生一个图像, 其中阿米巴和表面附着的细菌显示为深色区域 (图 3B)。</li>
	<li>确保在分析中检查区域和平均值的灰色值框|设置测量。通过在主工具栏上选择椭圆工具来测量阿米巴的近似大小。点击分析 |测量和注意代表阿米巴的面积。</li>
	<li>通过单击 "分析" 来创建阿米巴的掩码|分析粒子。在 "大小" 框中, 输入将包括阿米巴但排除细菌的区域。在 "放映" 选项的下拉菜单栏中选择 "掩码"。确保选中 "添加到管理器" 框。</li>
	<li>单击"确定" (OK ), 将显示仅显示阿米巴和 ROI 管理器对话框的图像 (图 3C)。</li>
	<li>使用文件打开荧光图像|打开并选择适当的文件。通过按住 Shift 键并单击列表中的每个区域, 选择ROI 管理器中的所有区域 (图 3D)。</li>
	<li>单击ROI 管理器中的 "度量" 按钮。这将打开一个结果窗口, 显示每个变形虫的平均强度值。</li>
	<li>将每个变形虫的荧光值复制到电子表格程序中, 并通过取所有荧光值的平均值来计算主机杀死指数。</li>
</ol>

<ol>
	<li>Rasko, D. A., Sperandio, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-virulence+strategies+to+combat+bacteria-mediated+disease.">Anti-virulence strategies to combat bacteria-mediated disease.</a> Nature Reviews Drug Discovery. 9 (2), 117-128 (2010).</li><li>Coburn, B., Grassl, G. A., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella,+the+host+and+disease:+a+brief+review.">Salmonella, the host and disease: a brief review.</a> Immunology and Cell Biology. 85 (2), 112-118 (2007).</li><li>Alibaud, 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=Pseudomonas+aeruginosa+virulence+genes+identified+in+a+Dictyostelium+host+model.">Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model.</a> Cellular Microbiology. 10 (3), 729-740 (2008).</li><li>Pukatzki, S., Kessin, R. H., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+pathogen+Pseudomonas+aeruginosa+utilizes+conserved+virulence+pathways+to+infect+the+social+amoeba+Dictyostelium+discoideum.">The human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum.</a> Proceedings of the National Academy of Sciences U S A. 99 (5), 3159-3164 (2002).</li><li>Cosson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+Virulence+Analyzed+in+a+Dictyostelium+discoideum+Host+System.">Pseudomonas aeruginosa Virulence Analyzed in a Dictyostelium discoideum Host System.</a> Journal of Bacteriology. 184 (11), 3027-3033 (2002).</li><li>Pukatzki, 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=Identification+of+a+conserved+bacterial+protein+secretion+system+in+Vibrio+cholerae+using+the+Dictyostelium+host+model+system.">Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system.</a> Proceedings of the National Academy of Sciences U S A. 103 (5), 1528-1533 (2006).</li><li>Steinert, M., Heuner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+as+host+model+for+pathogenesis.">Dictyostelium as host model for pathogenesis.</a> Cellular Microbiology. 7 (3), 307-314 (2005).</li><li>Hagele, S., Kohler, R., Merkert, H., Schleicher, M., Hacker, J., Steinert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+discoideum:+a+new+host+model+system+for+intracellular+pathogens+of+the+genus+Legionella.">Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella.</a> Cellular Microbiology. 2 (2), 165-171 (2000).</li><li>Matz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+uses+type+III+secretion+system+to+kill+biofilm-associated+amoebae.">Pseudomonas aeruginosa uses type III secretion system to kill biofilm-associated amoebae.</a> ISME Journal. 2 (8), 843-852 (2008).</li><li>Hauser, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+type+III+secretion+system+of+Pseudomonas+aeruginosa:+infection+by+injection.">The type III secretion system of Pseudomonas aeruginosa: infection by injection.</a> Nature Reviews Microbiology. 7 (9), 654-665 (2009).</li><li>Moore, P. L., MacCoubrey, I. C., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+pH+insensitive,+two+color+fluorescence+viability+(cytotoxicity)+assay.">A rapid pH insensitive, two color fluorescence viability (cytotoxicity) assay.</a> Journal of Cell Biology. 111, 58 (1990).</li><li>Kaneshiro, E. S., Wyder, M. A., Wu, Y. -. P., Cushion, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+calcein+acetoxy+methyl+ester+and+ethidium+homodimer+or+propidium+iodide+for+viability+assessment+of+microbes.">Reliability of calcein acetoxy methyl ester and ethidium homodimer or propidium iodide for viability assessment of microbes.</a> Journal of Microbiological Methods. 17 (1), 1-16 (1993).</li><li>Siryaporn, A., Kuchma, S. L., O'Toole, G. A., Gitai, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+attachment+induces+Pseudomonas+aeruginosa+virulence.">Surface attachment induces Pseudomonas aeruginosa virulence.</a> Proceedings of the National Academy of Sciences U S A. 111 (47), 16860-16865 (2014).</li><li>Decherchi, P., Cochard, P., Gauthier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+staining+assessment+of+Schwann+cell+viability+within+whole+peripheral+nerves+using+calcein-AM+and+ethidium+homodimer.">Dual staining assessment of Schwann cell viability within whole peripheral nerves using calcein-AM and ethidium homodimer.</a> Journal of Neuroscience Methods. 71 (2), 205-213 (1997).</li><li>Braut-Boucher, F., Pichon, J., Rat, P., Adolphe, M., Aubery, M., Font, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-isotopic,+highly+sensitive,+fluorimetric,+cell-cell+adhesion+microplate+assay+using+calcein+AM-labeled+lymphocytes.">A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.</a> Journal of Immunological Methods. 178 (1), 41-51 (1995).</li><li>Grieshaber, P., Lagr&#232;ze, W. A., Noack, C., Boehringer, D., Biermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+of+fluorogold-prelabeled+retinal+ganglion+cells+with+calcein-AM:+A+new+method+for+assessing+cell+vitality.">Staining of fluorogold-prelabeled retinal ganglion cells with calcein-AM: A new method for assessing cell vitality.</a> Journal of Neuroscience Methods. 192 (2), 233-239 (2010).</li><li>Witkin, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+Differences+in+Sensitivity+to+Radiation+in+Escherichia+Coli.">Inherited Differences in Sensitivity to Radiation in Escherichia Coli.</a> Proceedings of the National Academy of Sciences U S A. 32 (3), 59-68 (1946).</li><li>Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+Dictyostelium+discoideum+to+nucleic+acid+analogues.">Sensitivity of Dictyostelium discoideum to nucleic acid analogues.</a> Experimental Cell Research. 64 (2), 484-486 (1971).</li><li>Rahme, L. G., Stevens, E. J., Wolfort, S. F., Shao, J., Tompkins, R. G., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+virulence+factors+for+bacterial+pathogenicity+in+plants+and+animals.">Common virulence factors for bacterial pathogenicity in plants and animals.</a> Science. 268 (5219), 1899-1902 (1995).</li><li>O'Loughlin, C. T., Miller, L. C., Siryaporn, A., Drescher, K., Semmelhack, M. F., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quorum-sensing+inhibitor+blocks+Pseudomonas+aeruginosa+virulence+and+biofilm+formation.">A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm formation.</a> Proceedings of the National Academy of Sciences U S A. 110 (44), 17981-17986 (2013).</li></ol>

作者没有什么可透露的。

本协议描述一种快速、定量的方法来测定铜绿假单胞菌的毒力。此协议可与其他细菌进行测试。然而, 重要的是要记住, 生长培养基应与变形虫生长条件兼容。特别是, 我们已经优化的协议使用 PS: DB 作为细菌生长培养基。如果使用其他介质, 则可能需要执行仅生长介质控制, 在该控件中没有细菌细胞来验证介质是否与阿米巴兼容。
这一方法可以扩展, 以测定毒力的生长阶...

细菌感染是人类和动物死亡的主要原因之一,1,2。检测细菌在培养或生物膜中的毒力的能力在医疗保健和研究环境中很重要。在这里, 我们描述了一种通用的, 快速的, 相对简单的方法来量化细菌的毒性。以真核生物盘基柄柄(变形虫) 为模型宿主有机体。柄已被用作鉴定铜绿假单胞菌致病因子的宿主 (绿脓杆菌)3,4,5和其他细菌6,7 ,8 , 并且很容易受到同样的毒力因素的影响, 杀死哺乳动物细胞, 包括 III. 型分泌物9,10。以前使用柄的毒力测定, 在3、4、5小时的过程中, 会使细菌与d. 柄细胞长期接触。在这里, 该协议提出了一种快速的方法来确定毒力使用这种变形虫。本协议 (图 1) 描述了如何: (1) 生长阿米巴 axenically (在没有细菌的情况下), (2) 为化验培养细菌, (3) 为显微镜准备细菌和宿主细胞, (4) 执行荧光显微术, (5) 分析变形虫荧光。
阿米巴最初是从冰冻的储存和生长在大肠杆菌 (大肠杆菌) 的草坪上, 阿米巴产生孢子。这些孢子被采摘并接种成富含培养基无菌生长。阿米巴是通过无菌生长营养丰富的条件来维持的, 直到它们准备与细菌混合以评估细菌的毒力。阿米巴的生存或死亡通过测量 calcein-乙酰甲基 (calcein AM) 的荧光量量化, 这是由细胞内酯, 从而激活荧光11,12。活阿米巴表现很少或没有荧光, 而压力和垂死的细胞荧光强烈。这一结果是由于很少或没有纳入 calcein 的健康阿米巴和成立和分裂的基质在强调阿米巴13。这种行为明显有别于哺乳动物细胞11、14、15、16的 calcein 荧光。
将被评估为毒力的细菌分别生长。在这里, 我们描述如何测量机会病原菌的毒力, 并详细说明如何量化的毒性的浮游 (游泳) 和表面附着的亚人口。本议定书可用于测试其他细菌的毒力。在有代表性的结果部分, 我们表明, 毒力是激活的表面附着细胞和低的浮游细胞, 这是以前报告13。毒力活化的表面附着的铜绿假单胞菌杀死阿米巴, 而非毒性浮游细胞消耗的阿米巴。如果浮游细菌的毒力仅被测定, 细菌可以在普通的试管中培养, 而不是按照议定书中所描述的那样使用培养皿。
阿米巴和铜绿假单胞菌培养的生长必须协调, 以使铜绿假单胞菌培养达到预期的生长阶段, 而阿米巴在营养丰富的条件下生长在稳定的状态。这种情况通常要求阿米巴文化在与细菌混合之前至少1天被稀释。阿米巴和细菌是固定使用琼脂垫, 共孵化1小时, 并成像使用低分辨率 (10X, 数字光圈 0.3) 目标, 绿色荧光蛋白 (GFP) 过滤器, 和成像相机。可以使用自由可用的 ImageJ 软件或自定义图像分析软件进行分析。我们的分析是使用我们自己的软件编写的, 使用一个科学的分析包13。该软件应创建一个面具使用相对比图像和提取荧光值从蒙面区域的荧光图像。荧光值平均在至少100个细胞, 导致一个数字主机杀死指数。

<table border="0" cellpadding="0" cellspacing="0" style="width:716px;" width="715">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Reagents</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Bacto agar, dehydrated</td>
			<td style="width:108px;">BD Difco</td>
			<td style="width:91px;">214010</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Antibiotic-Antimycotic (100X)</td>
			<td style="width:108px;">Life Technologies</td>
			<td style="width:91px;">15240062</td>
			<td style="width:297px;">Aliquot &#60; 1 mL and store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Calcein-acetoxymethyl ester (calcein-AM)</td>
			<td style="width:108px;">Life Technologies</td>
			<td style="width:91px;">C34852</td>
			<td style="width:297px;">Calcein Acetoxymethyl (AM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Calcium chloride, anhydrous</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">C1016</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">D-Glucose</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">D16500</td>
			<td style="width:297px;">Dextrose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Dimethyl sulfoxide</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">D5879</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Folic acid</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">F8758</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">LB-Miller</td>
			<td style="width:108px;">BD Difco</td>
			<td style="width:91px;">244620</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Magnesium chloride</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">M8266</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Yeast extract</td>
			<td style="width:108px;">Oxoid</td>
			<td style="width:91px;">LP0021</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Special peptone</td>
			<td style="width:108px;">Oxoid</td>
			<td style="width:91px;">LP0072</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Potassium hyroxide</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">P250</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Potassium phosphate monobasic&#160;</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">P0662</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Sodium phosphate dibasic heptahydrate</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">S373</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Vitamin B12</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">V2876</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Strains</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:220px;">Dictyostelium discoideum</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">Strain AX318</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Escherichia coli</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">Strain B/r17</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Pseudomonas aeruginosa</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">PA14</td>
			<td style="width:297px;">PA14 strain19</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Pseudomonas aeruginosa &#916;lasR</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">AFS20.1</td>
			<td style="width:297px;">PA14-derived strain20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Supplies</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">0.22 &#181;m filter&#160;</td>
			<td style="width:108px;">Millipore</td>
			<td style="width:91px;">SCGPT01RE</td>
			<td style="width:297px;">For filter sterilization</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:220px;">Conical tube, 15 mL</td>
			<td style="width:108px;">Corning</td>
			<td style="width:91px;">352097</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Glass storage bottles</td>
			<td style="width:108px;">Pyrex</td>
			<td style="width:91px;">13951L</td>
			<td style="width:297px;">250 mL, 500 mL, 1000 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Petri dish, 6 cm diameter</td>
			<td style="width:108px;">Corning</td>
			<td style="width:91px;">351007</td>
			<td style="width:297px;">60 x 15 mm polystyrene plates</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:220px;">Petri dish, 10 cm diameter</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">FB0875712</td>
			<td style="width:297px;">100 x 15 mm polystyrene plates</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:220px;">Plastic containers with lid</td>
			<td style="width:108px;">Ziploc</td>
			<td style="width:91px;">2.57E+09</td>
			<td style="width:297px;">Square 3-cup containers</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:220px;">Glass plates</td>
			<td style="width:108px;">Bio-Rad</td>
			<td style="width:91px;">1653308</td>
			<td style="width:297px;">For preparing agar pads. Other glass plates may be used with similar dimensions.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Wooden sticks</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">23-400-102&#160;</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Equipment</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Eclipse Ti-E microscope&#160;</td>
			<td style="width:108px;">Nikon</td>
			<td style="width:91px;">MEA53100</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">10X Plan Fluor Ph1 objective&#160; 0.3 NA</td>
			<td style="width:108px;">Nikon</td>
			<td style="width:91px;">MRH20101</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Fluorescence excitation source</td>
			<td style="width:108px;">Lumencor</td>
			<td style="width:91px;">Sola light engine</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:220px;">Fluorescence filter set</td>
			<td style="width:108px;">Semrock</td>
			<td style="width:91px;">LED-DA/FI/TX-3X3M-A-000&#160;</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Orca Flash 4.0 V2 Camera</td>
			<td style="width:108px;">Hamamatsu</td>
			<td style="width:91px;">77054098</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">ImageJ</td>
			<td style="width:108px;">NIH</td>
			<td style="width:91px;">v. 1.49</td>
			<td style="width:297px;">Software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">MATLAB</td>
			<td style="width:108px;">Mathworks</td>
			<td style="width:91px;">R2013</td>
			<td style="width:297px;">Software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Orbital shaker incubator</td>
			<td style="width:108px;">VWR</td>
			<td style="width:91px;">89032-092</td>
			<td style="width:297px;">For growth of bacteria at 37 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Platform shaker</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">13-687-700</td>
			<td style="width:297px;">For growth of amoebae at 22 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Spectrophotometer</td>
			<td style="width:108px;">Biochrom</td>
			<td style="width:91px;">Ultrospec 10</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Undercounter refrigerated incubator</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">97990E</td>
			<td style="width:297px;">For growth of amoebae at 22 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Isotemp waterbath&#160;</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">15-462-21Q</td>
			<td style="width:297px;">For cooling media to 55 &#176;C</td>
		</tr>
	</tbody>
</table>

a rapid image-based bacterial virulence assay using amoeba

传统的细菌毒力检测包括在几个小时内长期暴露细菌, 以宿主细胞。在这段时间内, 由于接触宿主生长环境和宿主细胞的存在, 细菌可以经受生理变化。我们开发了一种快速测量细菌的毒力状态的检测方法, 以最大限度地减少细菌在宿主细胞存在时的生长程度。细菌和阿米巴混合在一起, 并固定在一个单一的成像平面上使用琼脂垫。该程序使用单细胞荧光成像与 calcein-乙酰甲基酯 (calcein) 作为一个指标的宿主细胞健康。利用荧光显微术对宿主细胞的1小时暴露后的宿主细胞进行荧光分析。图像分析软件用于计算主机的杀戮指数。该方法用于测定生物膜形成初期浮游和表面附着铜绿假单胞菌亚群中的毒力, 并可适应其他细菌和生物膜生长的其他阶段。该协议提供了一种快速和稳健的方法来测量毒力, 并避免了许多与生长和维护哺乳动物细胞系相关的复杂性。使用阿米巴测定的毒力表型也得到了使用小鼠巨噬细胞的验证。特别是, 这一检测被用来建立的表面附着上调毒力在铜绿假单胞菌。

我们在6厘米直径的培养皿中生长野生型铜绿假单胞菌 PA1419或ΔlasR菌株20 , 并测定浮游和表面附着细胞的毒力。文化从单一殖民地接种了入 PS: DB 文化, 在养殖管子隔夜生长在37°c 到饱和, 稀释1:100 入 ps: DB, 增长 8 h 在 6 cm 直径培养皿颤抖在100转, 并且浮游物和表面附上铜绿假单胞菌亚群被隔离, 如3节所述。
<p class="jo...

Watch this Scientific Journal Video about A Rapid Image-based Bacterial Virulence Assay Using Amoeba at JoVE.com

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

一种基于变形虫的快速图像细菌毒力检测方法

在这里, 我们提出了一个协议, 以测量浮游或表面附着细菌的毒力, 使用柄(变形虫) 作为宿主。在1小时内对毒力进行测量, 并使用荧光显微镜和图像分析对宿主杀死进行量化。我们用铜绿假单胞菌证明了这个协议。

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

a-rapid-image-based-bacterial-virulence-assay-using-amoeba

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.1K 次观看.  University of California. 在这里, 我们提出了一个协议, 以测量浮游或表面附着细菌的毒力, 使用柄(变形虫) 作为宿主。在1小时内对毒力进行测量, 并使用荧光显微镜和图像分析对宿主杀死进行量化。我们用铜绿假单胞菌证明了这个协议。

视频: A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

使用辅助电喷雾质谱（SESI - MS）表征细菌挥发

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

科研

生物工程

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

医疗器械的使用3D印刷模具的液体注射成型快速，低成本原型

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

测量TCR-的pMHC结合<em&gt;原位</em&gt;使用基于FRET的显微镜分析

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

一种快速和无农药血红蛋白检测法与光热角光散射

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

仿生纳米羟基磷灰石的快速混合制备生物医学应用

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

使用模块化金门克隆的多基因结构快速组装

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

通过交替粘性 - 惯性力喷射进行 体外 水凝胶微载体的3D打印

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

一种基于变形虫的快速图像细菌毒力检测方法

摘要

探索更多视频

此视频中的章节

使用辅助电喷雾质谱（SESI - MS）表征细菌挥发

使用电化学传感器的细菌检测和鉴定

医疗器械的使用3D印刷模具的液体注射成型快速，低成本原型

测量TCR-的pMHC结合

一种快速和无农药血红蛋白检测法与光热角光散射

仿生纳米羟基磷灰石的快速混合制备生物医学应用

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

使用模块化金门克隆的多基因结构快速组装

通过交替粘性 - 惯性力喷射进行体外水凝胶微载体的3D打印

一种基于变形虫的快速图像细菌毒力检测方法

摘要

探索更多视频

此视频中的章节

使用辅助电喷雾质谱（SESI - MS）表征细菌挥发

使用电化学传感器的细菌检测和鉴定

医疗器械的使用3D印刷模具的液体注射成型快速，低成本原型

测量TCR-的pMHC结合

一种快速和无农药血红蛋白检测法与光热角光散射

仿生纳米羟基磷灰石的快速混合制备生物医学应用

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

使用模块化金门克隆的多基因结构快速组装

通过交替粘性 - 惯性力喷射进行 体外 水凝胶微载体的3D打印

通过交替粘性 - 惯性力喷射进行体外水凝胶微载体的3D打印