多亏了旋律尼利, 乔恩艾伦, 巴塞尔 Abuaita, 唐娜 Runft, 他们努力帮助开发斑马鱼模型。这项研究报告得到了国家卫生研究院的国家过敏和传染性疾病研究所的支持, R21AI095520 和 R01AI127390 (对杰弗里 h. Withey) 的奖励数字。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。

这里描述的所有方法都得到了韦恩州立大学机构动物护理和使用委员会 (IACUC) 的批准。该方法首次在 Runft等中被描述。12
1. 肠道定植水平的测定
注: 肠道定植是斑马鱼模型中最有用的指标, 因为它可以用来比较各种v 型霍乱菌株的相对适用性或突变或基因挖空的影响。
<ol><li>利用浸泡法接种斑马鱼 注意: 这种接触方式类似于自然感染过程。<ol>
<li>霍乱弧菌的制备
		<ol>
<li>接种5毫升的溶源性汤 (磅) 与一个孤立的v 型霍乱菌群从 LB 板和孵化它在37°c 与震动 (180 rpm) 为 6–8 h (隔夜文化)。</li>
			<li>在250毫升锥形烧瓶中接种25毫升的新鲜蒸压 LB 培养基, 上面有50µL 的隔夜养殖。孵化它为 16–18 h 在37°c 与震动 (150 转每分钟)。</li>
			<li>阅读的光学密度在 600 nm (od600) 的一夜文化的1/10 稀释, 以估计细菌的数量每毫升 (OD600 = 1 是 ~ 109 CFU/毫升.)</li>
			<li>以6000克的离心方式收割细胞, 10 分钟. 用吸管取出介质或将其倒入消毒剂溶液中。并用重悬细胞在无菌 PBS 到期望集中, 通常在 1 x 107到 1 x 1010每毫升 PBS。 注: 在这里, 我们提到的蒸压反渗透水含有60毫克/升海盐作为无菌感染水。</li>
		</ol>
</li>
		<li>
接种和孵化
		<ol>
<li>将四或五斑马鱼放入400毫升烧杯中, 含200毫升无菌感染水。</li>
			<li>添加1毫升霍乱弧菌(从步骤 1.1.1.4) 获得所需的感染浓度 (通常为 5 x 104到 5 x 107 CFU/毫升) 在200毫升的感染水。</li>
			<li>用穿孔盖子盖上烧杯, 防止鱼跳出来;200µL 提示框的顶部工作正常。</li>
			<li>标签每个烧杯, 并把它们放到一个玻璃前孵化器设置在28摄氏度的试验期间。</li>
		</ol>
</li>
		<li>
暂时曝光程序 注: 暂时暴露于霍乱弧菌(通常为6小时) 之后, 除去接种细菌对研究传播和更准确地量化排泄细菌是有用的。<ol>
<li>经过6小时的曝光后, 通过渔网将烧杯中的水倒入收集鱼。将受感染的水丢弃到漂白容器中, 以杀死霍乱弧菌。</li>
			<li>将被去除的鱼放在200毫升无菌感染水的烧杯中。允许鱼在这干净的水中游泳5分钟, 以去除鱼表面的细菌。</li>
			<li>重复网络过程 (步骤 1.2.2), 并把鱼放在一个新的烧杯200毫升无菌感染水。在实验期间把鱼放在这个烧杯里。</li>
		</ol>
</li>
	</ol></li>
	<li>安乐 死
	<ol>
<li>当实验达到其预期的终点时, 取一个样本的感染水 (15 毫升通常是足够的), 以允许排泄细菌计数和测量腹泻 (如粘蛋白化验, 蛋白质含量和/或 OD), 如果需要 (2 节)。</li>
		<li>将剩余的水通过渔网 (收集鱼) 倒入漂白剂中, 以杀灭水中的霍乱弧菌。</li>
		<li>将鱼放入含有感染水的烧杯中, 再加336µg/毫升 3-氨基苯甲酸甲基磺酸乙酯 (tricaine), 并在该 tricaine 溶液中孵化出20分钟的鱼。 注: 罟用10% 漂白剂溶液消毒。</li>
	</ol></li>
	<li>夹层
	<ol>
<li>鱼的准备<ol>
<li>用一次性塑料勺子舀出 tricaine 溶液中的鱼, 将其放在解剖表面。</li>
			<li>将鱼与腹侧朝上, 将其钉在下颌上, 用针的钝端从身体中心处倾斜。把另一根针放在肛门后部, 也可以从身体角度离开。</li>
		</ol>
</li>
		<li>肠道暴露<ol>
<li>用70% 乙醇蘸上无绒布的擦拭, 拭去鱼腹面。</li>
			<li>用70% 乙醇蘸一把手术刀和 Vannas 剪刀, 然后将它们燃烧起来。</li>
			<li>通过穿透鳞片和使用手术刀, 在皮肤下面纵向切开一个小切口。小心不要切得太深, 因为肠道位于皮肤下面。</li>
			<li>使用剪刀, 沿着身体的长度仔细地伸展切口, 没有比皮肤更深的切割和避免肛门。</li>
			<li>用剪刀对鱼头进行两次横向切割, 使切口开口。</li>
			<li>将侧切口两侧的皮肤固定在解剖表面, 将针脚从身体上钓出来。 注意: 别针的尖端以前是通过酒精燃烧而消毒的。</li>
		</ol>
</li>
		<li>
肠道切除 注意: 肠道应该是可见的, 在其他器官之上的苍白, 非常薄的管。<ol>
<li>用火焰消毒镊子, 然后用它们去除整个肠道 (通常12–15毫米)。将肠道放入含有玻璃珠的均匀化管 (见步骤 1.4. 1–1.4 2) 和1毫升 1x PBS 或磅, 在冰上。</li>
		</ol>
</li>
	</ol></li>
	<li>均匀
	<ol>
<li>先将1.5 克1毫米玻璃珠倒入2毫升螺帽管 (大约半路), 然后用热处理消毒。</li>
		<li>添加1毫升不育 LB 或 1x PBS。</li>
		<li>一旦斑马鱼的肠道被添加 (步骤 1.3.3.1) 到管, 拧紧的上限, 然后确保在漩涡均质管。</li>
		<li>融汇1分钟的样品在最大的设置, 然后冷却他们在冰上的最小. 重复此均匀化循环一次, 以完全均匀化。 注意: 几种不同的均匀化方法也会奏效。</li>
	</ol></li>
	<li>定量肠道定植水平
	<ol>
<li>在每管中加入900µL 的 LB 或 1x PBS, 准备管子 (小玻璃试管或1.5 毫升离心管), 用于连续稀释匀浆。为每条鱼, 准备5–6管和做10倍的肠道匀浆稀释, 添加100µL 的匀浆到第一管, 涡流它混合, 然后增加100µL 从第一管到第二管。</li>
		<li>重复此过程, 直到所有稀释都已准备就绪。</li>
		<li>在含有100µg/毫升链霉素 (如果使用链霉素抗性菌株) 和40µg/毫升 X-加仑 (5-溴-4-氯-3-哚β D-galactopyranoside) 的 LB 琼脂板上的每稀释100–200µL。在30摄氏度孵化出 16–18 h 的盘子。</li>
		<li>在夜间孵化后, 在盘子上数诉霍乱菌落, 或者使用自动菌落计数器或手动计数菌落;用标记尖标记计数的殖民地是有用的。</li>
		<li>在考虑到所镀的体积的情况下, 用悬浮液的稀释系数乘以板数, 确定每条鱼肠的 CFU。</li>
	</ol></li>
</ol>2. 鱼腹泻的测量
注: 四种不同的指标用于评估鱼腹泻: 水中的黏蛋白水平、水中的总排泄蛋白水平、水的 OD600和霍乱弧菌在水中的 CFU13。如上文所述, 斑马鱼接触到霍乱弧菌后, 腹泻通常在视觉上明显接近6小时。
<ol><li>
确定黏蛋白水平 注: 粘液分泌在霍乱弧菌定植期间诱发, 是一种很好的排泄水平, 尤其是早期感染13。粘蛋白测定是微量滴定周期酸性席夫碱测定19的一种变异。<ol>
<li>准备以下材料: 50% (w/v) 周期性酸库存溶液和0.1% 周期酸工作溶液 (10 µL 50% 周期性酸库存添加到5毫升的 7% mL 乙酸-立即使用后), 黏蛋白标准, 96 井微量滴定板, 希夫的试剂。<ol>
<li>通过悬浮以下数量的黏蛋白 (从猪胃 III.) 在乙酸钠缓冲液 (100 毫米醋酸钠, 5mM EDTA, pH 值到5.5 与冰川乙酸) 制备粘蛋白标准: 400 µg/毫升, 300 µg/毫升, 200 µg/毫升, 150 µg/毫升, 100 µg/毫升, 75 µg/毫升, 50 µg/毫升, 25 µg/毫升, 10 µg/毫升。 将100µL 的感染水添加到每一个井中, 并将其用于化验, 如下所示: 空白 1x PBS;如上文所述的黏蛋白标准, 或从鱼类感染中抽取水样。每样样品三份。</li>
		</ol>
</li>
		<li>添加50µL 新鲜0.1% 周期酸溶液, 每井与多通道吸管, 并混合它由吹打上下几次。</li>
		<li>把盘子紧紧地裹在塑料包里, 在37摄氏度的 1–1.5 h 上孵化。</li>
		<li>把盘子冷却到室温, 5–10最小. 添加100µL 的品红溶液 (希夫的试剂) 给每个井, 并吸管上下混合。</li>
		<li>包装在塑料包装板, 并在室温下孵化它在一个摇摆平台, 直到颜色已经发展;颜色一般是在20分钟内开发的. 用平板阅读器读取 560 nm 的吸光度。</li>
		<li>绘制一个黏蛋白标准曲线 (OD560与µg/毫升粘蛋白标准)。通过确定每个未知的 OD560在标准曲线上的位置, 并读取该值的相应黏蛋白浓度, 确定未知的黏蛋白水平。</li>
	</ol>
</li>
	<li>
确定蛋白质水平 注: 除粘蛋白外, 水中的总蛋白质水平是鱼类产生的腹泻 (混浊、液体大便) 水平的另一个代表。本程序使用标准的布拉德福德化验来估计蛋白质水平。蛋白质水平估计根据牛血清白蛋白 (BSA) 标准曲线。<ol>
<li>混合100µL 的 BSA 标准之一 (见下文) 或100µL 感染水 (含受感染鱼类的烧杯中的水) 与900µL 的布拉德福德化验试剂 (皮尔斯蛋白化验试剂工作很好, 这) 在一次性试管。在室温下孵化所有样品2分钟。 注: 使用了以下 BSA 标准: 1800 µg/毫升, 1000 µg/毫升, 750 µg/毫升, 500 µg/毫升, 250 µg/毫升, 125 µg/毫升, 50 µg/毫升, 25 µg/毫升。在蒸压蒸馏水中, BSA 标准被稀释。</li>
		<li>阅读每个标准的 OD660或使用分光光度计取样。</li>
		<li>绘制 BSA 标准曲线 (OD660与µg/毫升)。确定未知的蛋白质水平, 通过确定每个未知的 OD660落在 BSA 标准曲线上, 并读取该值的相应粘蛋白浓度。</li>
	</ol>
</li>
	<li>
测量 OD600 注: 腹泻 (混浊, 液体大便) 的存在明显明显后, 几个 h 和一个简单的 OD600确定可以用来量化这一点。<ol>
<li>将含感染水的管倒置数次, 均匀分布。将1毫升的水样本添加到一次性试管。以1毫升无菌感染水为阴性对照。</li>
		<li>使用负控制样本, 使用 600 nm 的分光光度计执行 "空白" 测量。阅读每个水样600毫微米和记录的结果。</li>
	</ol>
</li>
	<li>
感染后 CFU霍乱弧菌排泄量的测定 注: 斑马鱼产生的腹泻的一个主要成分是排出霍乱弧菌。CFU/mL 的测定可用于评估鱼类肠道细菌复制, 以及作为传播实验中传染性剂量的量度。这项措施最好是在发生短暂感染时进行的。如果使用 24 h 感染, 则此检测将测量联合接种加排出的霍乱弧菌。<ol>
<li>在所需的时间点取一个水样, 并按前面所描述的顺序稀释它。</li>
		<li>在选择性培养基上印上系列稀释 (含有链霉素的 LB 琼脂或另一种适当的抗生素或 DCLS), 并将其孵化为24小时30摄氏度。</li>
		<li>数殖民地。计算步骤1.5 中描述的每毫升水的 CFU。</li>
	</ol>
</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+adult+rabbit+model+for+Vibrio+cholerae+and+enterotoxigenic+Escherichia+coli+diarrhea.">Simple adult rabbit model for Vibrio cholerae and enterotoxigenic Escherichia coli diarrhea.</a> Infection and Immunity. 32 (2), 739-747 (1981).</li><li>Ritchie, J. M., Rui, H., Bronson, R. T., Waldor, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Back+to+the+future:+studying+cholera+pathogenesis+using+infant+rabbits.">Back to the future: studying cholera pathogenesis using infant rabbits.</a> mBio. 1 (1), (2010).</li><li>Kilcoyne, M., Gerlach, J. Q., Farrell, M. P., Bhavanandan, V. P., Joshi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodic+acid-Schiff's+reagent+assay+for+carbohydrates+in+a+microtiter+plate+format.">Periodic acid-Schiff's reagent assay for carbohydrates in a microtiter plate format.</a> Analytical Biochemistry. 416 (1), 18-26 (2011).</li><li>Balaji, V., Sridharan, G., Jesudason, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+of+non+O1,+non+O139+Vibrios+isolated+from+fresh+water+bodies+in+Vellore,+south+India.">Cytotoxicity of non O1, non O139 Vibrios isolated from fresh water bodies in Vellore, south India.</a> Indian Journal of Medical Research. 110, 155-159 (1999).</li><li>Hasan, N. A., 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=Nontoxigenic+Vibrio+cholerae+non-O1/O139+isolate+from+a+case+of+human+gastroenteritis+in+the+U.S.+Gulf+Coast.">Nontoxigenic Vibrio cholerae non-O1/O139 isolate from a case of human gastroenteritis in the U.S. Gulf Coast.</a> Journal of Clinical Microbiology. 53 (1), 9-14 (2015).</li></ol>

作者没有什么可透露的。

斑马鱼是研究霍乱弧菌的一种比较新的模式, 但对于今后发现霍乱弧菌生物学和发病机制11、12、13等未知方面有很大希望。.成年斑马鱼模型具有天然v 型霍乱宿主的优势, 包括完整的、成熟的肠道微生物群和环境模型。该模型的缺点是, 两个主要的人类毒力因素, 霍乱毒素和毒素-coregulated pilus, 不需要为斑马?...

霍乱弧菌是一种水生, 革兰阴性菌, 导致人类疾病霍乱以及零星腹泻1,2。霍乱是在全球许多地区的环境中发现的, 通常与其他水生生物有关。这些关联有机体包括浮游生物, 昆虫蛋大量, 贝类和脊椎动物种类3,4,5,6,7。有几项研究从7、8、9、10不同地理区域的鱼类肠道中分离出霍乱弧菌。在鱼类中存在霍乱弧菌, 表明鱼可能充当环境水库。鱼类还可能牵涉到向人类传播这种疾病, 并在地理上传播霍乱6株。
为了更好地了解霍乱弧菌与鱼类的相互作用,斑马斑马, 更被称为斑马鱼, 是研究霍乱e11的模型系统。斑马鱼原产于南亚, 包括孟加拉湾地区, 被认为是霍乱弧菌最早的水库。在1817年开始的第一次霍乱大流行之前, 除了目前的印度和孟加拉国以外, 没有报告霍乱。因此, 斑马鱼和霍乱弧菌在进化时间尺度上几乎肯定是相互关联的, 这表明斑马鱼是12自然环境中的五个霍乱弧菌宿主。
斑马鱼模型为霍乱弧菌是简单的执行和可用于研究整个致病性霍乱弧菌生命周期。鱼通过沐浴在已接种了已知数量的霍乱弧菌的水中, 接触到霍乱弧菌。在几个小时内, 肠道定植发生, 其次是腹泻的产生。腹泻包括粘液、蛋白质、排泄细菌和其他肠道内容。腹泻的程度可以用一些简单的测量13来量化。被感染的鱼排出的霍乱弧菌可以继续感染天真的鱼, 完成感染周期。因此, 斑马鱼模型概括 v.12、14的霍乱弧菌。
最常用的v. 霍乱动物模型历来是老鼠和兔子14,15,16,17,18。这些模型有助于增加我们对霍乱病发病机制的认识。然而, 由于老鼠和兔子不是天然的v. 霍乱弧菌宿主, 因此, 对霍乱弧菌生命周期的哪些方面可以进行研究。霍乱弧菌对小鼠和家兔的定植通常要求缺乏肠道微生物群或用抗生素进行预处理, 以损害肠道微生物群。两种模型都要求饲将细菌引入消化道或手术操作, 直接将细菌注入肠道。斑马鱼有一个优势, 即成年鱼类与完整的肠道微生物群容易被殖民和感染过程自然发生, 没有任何操作需要。
目前的研究表明, 斑马鱼作为霍乱弧菌感染模型的应用。12、13, 对霍乱弧菌的感染、解剖、计数, 以及霍乱弧菌引起的腹泻的量化进行了说明。这种模式可能对那些对霍乱弧菌疾病过程和霍乱弧菌的环境生活方式感兴趣的科学家有用。

<table>
	<tbody>
		<tr>
			<td>Instrument</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker incubator</td>
			<td>New Brunswick Scientific, Edison, NJ</td>
			<td>Excella E25</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>NUAIRE, Plymouth, MN</td>
			<td>Auto Flow</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo, Waltham, MA</td>
			<td>Geaesys 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex homogenizer</td>
			<td>Minibeadbeater24</td>
			<td>112011</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing Machine</td>
			<td>Ohaus, Columbia, MD</td>
			<td>Adventurer Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Stirer</td>
			<td>Corning, Corning, NY</td>
			<td>PC-420D</td>
			<td></td>
		</tr>
		<tr>
			<td>Burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>automated colony counter</td>
			<td>REVSCI</td>
			<td>120417B</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>400 ml glass beakers</td>
			<td>Pyrex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>perforated lids</td>
			<td>Microtip holder with holes from tip box</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>disposable plastic spoons</td>
			<td>Office Depot, Boca Raton, FL</td>
			<td>D15-25-7008</td>
			<td></td>
		</tr>
		<tr>
			<td>Fish Tank System</td>
			<td>Aquaneering, San Diego, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RO Water Purifier</td>
			<td>Aqua FX</td>
			<td>TK001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fish net</td>
			<td>Marina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fish food</td>
			<td>Tetra fin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brine Shrimp</td>
			<td>Red jungle brand</td>
			<td>O.S.I. pro 80</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam board</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Fine Scientific tools, Foster City, CA</td>
			<td>10000-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Scientific tools, Foster City, CA</td>
			<td>11223-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas scissors</td>
			<td>Fine Scientific tools, Foster City, CA</td>
			<td>15000-11</td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml screw cap tubes</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>02-681-375</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mm glass beads</td>
			<td>Bio Spec</td>
			<td>11079110</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads for spreading</td>
			<td>Sigma, St. Louis, MO</td>
			<td>18406-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri plate</td>
			<td>Fisher Brand, Hampton, NH</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml centrifuge tube</td>
			<td>Midsci, Valley Park, MO</td>
			<td>AVSS1700</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>Corning Falcon, Corning, NY</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Test tubes</td>
			<td>Pyrex</td>
			<td>9820</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pipette</td>
			<td>Fisher Brand, Hampton, NH</td>
			<td>13675K</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pipettes</td>
			<td>Sartorius Biohit, G&#246;ttingen, Germany</td>
			<td>m1000/m200/m20</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips</td>
			<td>Genesee Scientific, San Diego, CA</td>
			<td>24-150RS/24-412</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Ocean salts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>VWR Life Science, Radnor, PA</td>
			<td>K813-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td>Sigma, St. Louis, MO</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside</td>
			<td>Sigma, St. Louis, MO</td>
			<td>10651745001</td>
			<td></td>
		</tr>
		<tr>
			<td>Schiff&#8217;s reagent</td>
			<td>Sigma, St. Louis, MO</td>
			<td>84655-250 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>periodic acid</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>10450-60-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucin from porcine stomach</td>
			<td>Sigma, St. Louis, MO</td>
			<td>M2378-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>9046-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 660nm Protein Assay Reagent</td>
			<td>Thermo, Waltham, MA</td>
			<td>22660</td>
			<td></td>
		</tr>
		<tr>
			<td>LB medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypton</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Teast Extract</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>NACL</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>TCBS Agar</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td>265020</td>
			<td></td>
		</tr>
		<tr>
			<td>DCLS Agar</td>
			<td>Sigma, St. Louis, MO</td>
			<td>70135-500gm</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft office</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying vibrio cholerae colonization and diarrhea in the adult zebrafish model

霍乱弧菌是最有名的感染性药物, 引起了人类疾病。在人类宿主之外,霍乱弧菌主要存在于水生环境中, 在那里它与各种更高的水生物种相互作用。众所周知, 脊椎动物是一种环境宿主, 是一种潜在的霍乱弧菌储层。霍乱弧菌和硬骨鱼类鱼类种类斑马斑马, 俗称斑马鱼, 起源于印度次大陆, 表明在水生环境中存在长期的相互作用。斑马鱼是研究生物的许多方面的理想的模型有机体, 包括传染性疾病。在水中暴露后, 斑马鱼可以很容易和迅速地被霍乱弧菌所殖民。霍乱弧菌引起的肠道定植导致腹泻的产生和复制的霍乱弧菌的排泄。这些排泄出来的细菌可以继续殖民新的鱼类寄主。在这里, 我们示范如何评估v.斑马鱼的霍乱-肠道殖民化, 以及如何量化五、霍乱引起的斑马鱼腹泻。殖民模式应该是有用的研究人员谁正在研究是否有兴趣的基因可能是重要的宿主殖民和/或对环境的生存。对于研究任何有兴趣探索斑马鱼作为模型系统的肠道病原体的研究人员来说, 斑马鱼腹泻的量化应该是有用的。

五、霍乱斑马鱼肠道的定植
为了提供我们观察到的典型的殖民化水平的例子, 我们接种了 5 x 106 CFU 的大流行性流感病毒菌株 N16961 在装有几只斑马鱼的烧杯中的200毫升水中。经过6小时的感染, 这些鱼被淡水洗涤, 并转移到一个烧杯200毫升的蒸压感染水, 如议定书所述。18 h 在转移之后 (或 24 h 在主要传染以后),...

Watch this Scientific Journal Video about Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model at JoVE.com

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

斑马鱼是一种天然的霍乱弧菌宿主, 可以用来重述和研究整个感染周期从殖民化到传播。在这里, 我们演示如何评估霍乱弧菌的殖民水平和量化腹泻的斑马鱼。

成年斑马鱼模型中霍乱弧菌定植与腹泻的量化研究

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

quantifying-vibrio-cholerae-colonization-diarrhea-adult-zebrafish

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Immunology and Infection

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

8.3K 次观看.  Wayne State University School of Medicine. 斑马鱼是一种天然的霍乱弧菌宿主, 可以用来重述和研究整个感染周期从殖民化到传播。在这里, 我们演示如何评估霍乱弧菌的殖民水平和量化腹泻的斑马鱼。

视频: Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

在无菌无鼠标进定植评估肝代谢的变化 1 H核磁共振光谱

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

科研

免疫与感染

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

定植 Euprymna scolopes鱿鱼由弧菌鲵</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

TransFLP - A基因修改方法<em&gt;霍乱弧菌</em基于自然转化和FLP重组

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from &#945;-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.

正常和恶性肌细胞移植到免疫受损成年斑马鱼

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

对于检测到的敏感性和特异性显色琼脂培养基发展<em&gt;副溶血性弧菌</em&gt;及其他<em&gt;弧菌</em&gt;物种

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

用于维持和区分生物型的实验室技术<em&gt;霍乱弧菌</em&gt;临床和环境隔离

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

成年斑马鱼模型中霍乱弧菌定植与腹泻的量化研究

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在无菌无鼠标进定植评估肝代谢的变化¹ H核磁共振光谱

定植 Euprymna scolopes鱿鱼由弧菌鲵

TransFLP - A基因修改方法

培养小胶质细胞的新生和成年的中枢神经系统

使用一个调查益生素对肺炎球菌定植的影响

正常和恶性肌细胞移植到免疫受损成年斑马鱼

对于检测到的敏感性和特异性显色琼脂培养基发展

B组的小鼠模型

用于维持和区分生物型的实验室技术

人单核细胞趋化体外和小鼠淋巴细胞贩运体内