我们感谢尼朋·巴斯鲁尔、阿德里亚娜·罗萨斯·维尔加斯、纳达夫·沙伊和特雷弗·索雷尔对手稿的评论，以及宫中燕和基罗洛斯·巴苏姆的技术援助。我们感谢亚历克斯·威尔德在 图1中使用的照片。K.V.得到了博林格·英格尔海姆基金会博士奖学金的支持。V.J.部分得到NIH T32-MH095246的支持。这项工作的部分支持来自洛克菲勒大学从霍华德休斯医学研究所通过詹姆斯H.吉利亚姆高级研究奖学金计划到V.J.该材料基于国家科学基金会研究生研究奖学金计划支持的工作，该奖学金项目由第1号赠款公司提供。NSF DGE-1325261 到 V.J.本材料中表达的任何意见、发现、结论或建议均为作者的意见，不一定反映国家科学基金会的观点。

血液喂养程序不使用活体动物或人类宿主进行，并符合洛克菲勒大学机构动物护理和使用委员会（IACUC）和机构审查委员会（IRB）制定的准则。
1. 膳食准备
<ol><li>法戈刺激剂，腺苷5+三磷酸酯的制备<ol>
<li>准备25米水性NaHCO3 溶液（分子量，兆瓦=84.006克/摩尔）。对于 100 mL 的 25 mM NaHCO3，将 210 毫克 NaHCO3 添加到体积烧瓶中，并填充双蒸馏水（ddH2O），总体积为 100 mL。使用磁搅拌棒，彻底混合溶液，直到所有 NaHCO3 溶解。</li>
		<li>在水性 25 mM NaHCO3 中重组 ATP 脱钠盐水合物 （MW = 551.14 g/mol），最终浓度为 200 mM ATP。对于总体积为 10 mL 的 200 mM ATP 在 25 mM NaHCO3 缓冲区，将 1.1 克 ATP 脱钠盐水合物添加到体积瓶中，并填充 25 mM NaHCO3 缓冲器，总体积为 10 mL。使用磁搅拌棒，彻底混合溶液，直到所有 ATP 溶解。 注：要尽量减少ATP的水解，必须通过纳赫科3等盐溶液进行缓冲。</li>
		<li>阿里引用ATP解决方案，并在-20°C存储。 注：ATP的这种库存解决方案通常每六个月新鲜一次，用于下面描述的所有膳食。为了防止退化，ATP 别名不应经历多个冷冻解冻周期，也不应与其他膳食组件一起加热。</li>
	</ol></li>
	<li>荧光示踪剂溶液、荧光素的制备<ol>
<li>准备2%（w/v）的液态荧光素库存溶液。对于总库存溶液量为 10 mL，在室温下用铝箔包裹的 15 mL 锥形管中混合 0.2 克荧光素脱钠盐和 10 mL 的 ddH2O。这种荧光素的库存溶液可用于以下所有膳食的稀释。 注：由于荧光素对光敏感，请用铝箔包装容器，避免暴露在光线下。</li>
	</ol></li>
	<li>准备动物源性血液餐<ol>
<li>计算喂养所有蚊子所需的膳食数量;每个格利图伯有一顿2mL的饭菜，喂养大约25只蚊子。准备一顿额外的膳食，以校准荧光读数的标准曲线。除非另有说明，否则本节中的所有步骤都描述了准备一顿饭所需的试剂量，最终体积为 2 mL。</li>
		<li>对于动物源性血液餐，将 1.98-2 mL 除颤羊血转移到 15 mL 锥形管中（参见步骤 3.3，以获得所需的血液量）。 注：商业去除脊椎动物血液来源，包括绵羊、豚鼠和人类，可使用13。使用前，确保所购血液未过期，并通过倒置瓶子将其混合好，尤其是在血液成分明显分离的情况下。</li>
		<li>为了获得最佳喂养，在水浴中将羊血加热到 45 °C 后，将 ATP 添加到 1-2 mM 的最终浓度中。对于 1 mM ATP 的最终浓度，在 200 mM ATP 库存解决方案中添加 10 μL，加入 1.99 mL 的预热血和混合液。对于 2 mM ATP 的最终浓度，将 200 mM ATP 库存中的 20 μL 添加到 1.98 mL 的预温血液和混合物中。如果不添加 ATP，加热 2 mL 的除颤羊血。</li>
		<li>如果随后要对膳食大小进行基于荧光的量化，则将荧光素溶液添加到最终浓度为 0.002% （2 μL 的 2% 荧光素库存在 2 mL 总膳食量中）。将血液量减少与添加的荧光素相同的量。保留含有 0.002% 荧光素的最终膳食配方的 1 mL 以生成参考标准曲线。将保留的体积与提供给蚊子的膳食相同;在整个实验过程中暴露在相同的光线和温度条件下，然后与送餐一起冷冻。</li>
	</ol></li>
	<li>制备人造血餐<ol>
<li>计算喂养所有蚊子所需的膳食数量;每个格利图伯有一顿2mL的饭菜，喂养大约25只蚊子。准备一顿额外的膳食，以校准荧光读数的标准曲线。除非另有说明，否则本节中的所有步骤都描述了准备一顿 2 mL 餐所需的试剂量。</li>
		<li>为了准备人造血液（改编自科根（1990年）22），如 表1，首先使库存溶液400 mM NaHCO3。 总容量为 10 mL 的 400 mM NaHCO3 （MW = 84.006 g/mol），将 336 毫克 NaHCO3 添加到体积烧瓶中，并填充双蒸馏水（ddH2O），总体积为 10 mL。使用磁搅拌棒，彻底混合溶液，直到所有 NaHCO3 溶解。</li>
		<li>对于人造血液的蛋白质成分，在400 mM NaHCO3中制备50毫克/毫升γ球蛋白、在ddH2O中35毫克/mL的血红蛋白和在ddH2O中300毫克/毫升的白蛋白的库存溶液，蛋白质库存溶液可在4°C储存长达2个月。人造血液中人类蛋白质总量的最终浓度为125毫克/mL。这包括15毫克/mL γ球蛋白、8毫克/mL血红蛋白和102毫克/mL白蛋白的最终浓度。</li>
		<li>每2毫升餐，结合600μL的γ球蛋白，460μL的血红蛋白，680μL的白蛋白，和250μL的ddH2O从 表1中列出的股票解决方案。等待添加 10μL 的 200 mM ATP 库存解决方案，直到饭后加热到 45 °C，然后立即呈现餐食。</li>
		<li>如果随后要对膳食大小进行基于荧光的量化，则将荧光素溶液添加到最终浓度为 0.002% （2 μL 的 2% 荧光素库存在 2 mL 总膳食量中）。在步骤4.4中减少 ddH 2 O 的体积，其量与添加的荧光素相同。保留含有0.002%荧光素的最终膳食配方至少1 mL以生成参考标准曲线。将保留的体积与提供给蚊子的膳食相同;在整个实验过程中暴露在相同的光线和温度条件下，然后与送餐一起冷冻。</li>
	</ol></li>
	<li>准备无蛋白盐水餐（改编自杜瓦尔等人（2019）7） 注：无蛋白质盐水餐可以以多种方式准备7，27，32。这里介绍的盐水餐是上述人工血液配方的无蛋白质版本。<ol>
<li>计算喂养所有蚊子所需的膳食数量;每个 Glytube 都提供 2 mL 餐食，并喂养大约 25 只蚊子，准备一顿额外的膳食，以校准荧光测量的标准曲线。除非另有说明，否则本节中的所有步骤都描述了准备一顿 2 mL 餐所需的试剂量。</li>
		<li>要准备盐水餐，制作 400 mM NaHCO3的库存溶液。对于总体积为 10 mL 的 400 mM NaHCO3 （MW = 84.006 g/mol），将 336 毫克的 NaHCO3 添加到体积烧瓶中，并填充 ddH2O，总体积为 10 mL。使用磁搅拌棒，彻底混合溶液，直到所有 NaHCO3 溶解。</li>
		<li>对于每2毫升餐，结合在一个15毫升锥形管600μL的400 mM NaHCO3 与1.39毫升的ddH2O.等待添加10μL的200 mM ATP库存解决方案，直到饭后已加热到45°C在水浴。</li>
		<li>如果随后要对膳食大小进行基于荧光的量化，则将荧光素溶液添加到最终浓度为 0.002% （2 μL 的 2% 荧光素库存在总膳食量的 2 mL 中）。在步骤5.3中减少 ddH 2 O 的体积，其量与添加的荧光素相同。保留含有0.002%荧光素的最终膳食配方至少1 mL以生成参考标准曲线。将保留的体积与提供给蚊子的膳食相同;在整个实验过程中暴露在相同的光线和温度条件下，然后与送餐一起冷冻。</li>
	</ol></li>
</ol>2. 送餐给蚊子
<ol><li>设置供喂养的蚊子容器 注：只要符合以下标准，蚊子就可以在各种容器中喂养。确保容器足够大，蚊子可以飞来飞去，但不要太大，蚊子很难找到网状表面并开始进食。用于覆盖容器的网格在材料和孔大小上可能有所不同。孔必须足够大，雌性蚊子的风格穿透，但不是那么大，蚊子可以逃脱。牢牢地固定网格，使其绷紧，Glytube 在整个喂食期间可以稳定地放在其表面上。<ol>
<li>例如容器 （图 1）是一个经过改装的 946 mL （32 盎司） 高密度聚乙烯 （HDPE） 塑料桶。要复制此设置，请使用剃须刀刀片在桶盖中切割直径约 10 厘米的中央孔。要组装容器供蚊子占据，在桶顶上固定一块约 400 厘米2 平方的白色 0.8 毫米聚酯蚊帐，将穿孔盖牢牢地推倒在水桶上，以紧握。</li>
		<li>收集关闭后至少3天的雌性蚊子，以确保它们足够成熟，可以喂血。最佳喂养率在7天33后观察到。</li>
		<li>将雌性蚊子放入容器中，并盖上网状。如果容器中密布蚊子，则增加使用的胶合管数量。最佳喂养是通过+25蚊子/胶管实现的。这减少了获得喂食膜的竞争。</li>
		<li>留出一组未受控制的蚊子，这些蚊子不会得到一顿饭。在体重测量协议中，分别对未受约束的组进行称重，并使用此重量来估计以膳食为食的实验组的体重增加。在基于荧光的量化协议中，将未受约束的蚊子群添加到井中，用于标准曲线计算和负控制。为了匹配实验组的基线蚊子组织自荧光，确保标准曲线和负控制井包含未受蚊子。</li>
	</ol></li>
	<li>建造和建立格利图布 （改编自科斯塔 - 达西尔瓦等人 （2013）20）<ol>
<li>如 图 1所示，要生成热源，请将 50 mL 锥形管填充 40 mL 的 100% 甘油。用 5 厘米× 5 厘米的护膜密封打开的圆锥管，再加一块 5 厘米× 5 厘米的护膜，以最大限度地减少泄漏的机会。可选地，可以使用橡皮筋将准胶片放在原位。倒置管，以确保没有孔或缝隙。</li>
		<li>要创建送餐装置，只需使用锋利的剃须刀刀片或车床，在圆锥管螺钉盖上切割一个直径为 2.5 厘米的中心孔。均匀地伸展一块 5 厘米× 5 厘米的准胶片，使其大小大致翻倍。准胶片应该足够薄，蚊子很容易穿透它，但不应该有泄漏。密封螺钉盖的外表面，以完全覆盖孔，并设置盖子一边。 注意：为了增加对Glytube的吸引力，在拉伸护膜之前，用人类的气味来香水，轻轻揉在一块没有化妆品的人体皮肤上，注意不制造任何孔洞。如果实验的目的不是调查蚊子接近膳食所需的感官线索，建议这样做。</li>
		<li>在42-45°C的水浴中加热甘油和膳食的密封管（除ATP外的所有组件），至少15分钟。不要预热ATP：在开始实验前立即添加它。</li>
		<li>将 ATP 添加到加热的膳食中，并彻底转到漩涡中。移液器 2 mL 的加热餐进入螺丝帽的内室，轻轻地将倒置、加热、甘油填充的 50 mL 锥形管放入螺丝帽中。部分拧盖与膳食甘油填充管-只是足以防止泄漏的膳食或甘油。 注意：使用的膳食量在1 mL和2.5 mL之间。当餐食用于提供稀缺或昂贵的化合物时，较低的体积可能特别有用。在此步骤中快速工作非常重要，以便膳食不会冷却到环境温度，并降低最大进食的可能性。冷却速度将取决于执行这些步骤的房间的环境温度，但通常应在 5 分钟内在 25 °C 完成。</li>
		<li>将组装好的 Glytube 放在蚊子容器的顶部，让蚊子至少食用 15 分钟，以达到最大喂养率。</li>
		<li>为了获得最佳的喂养，请将蚊子容器放置在装有 CO2 垫的室内，并在送餐前在 25-28 °C 和 70-80% 的湿度下进行至少 15 分钟的适应。这里使用的检测室是一个简单和低成本的修改，以前公布的设置16。它使用半透明聚丙烯存储盒大小 36 厘米 L × 31 厘米 W × 32 厘米 H 与可拆卸盖。室壁上直径为 1.5 厘米的孔允许通过硅胶管输送 CO2。 CO2 扩散垫贴在盖子的内侧中心，用于输送净化空气和 CO2， 以在试验期间调节室内气氛。 注意：确保宿主提示（热和二氧化碳，可选宿主气味16）存在，以便蚊子被吸引到膜喂食器。如果蚊子没有挤在 Glytube 下面，请检查 CO2 是否正确传递，膳食和 Glytube 是否足够温暖。如果外部 CO2 源不可用，CO2 可以通过呼出的人类呼吸气泡传递。</li>
		<li>喂养后，Glytube 帽可以作为生物危害废物丢弃，或浸泡在低百分比漂白液中并彻底冲洗在水中后再使用。</li>
	</ol></li>
</ol>3. 消费膳食的量化
<ol><li>称量蚊子用于进一步实验 注：称量蚊子以量化膳食大小允许它们用于进一步的活体实验，但这种方法需要从一组5只蚊子中测量体重。由于使用大多数实验室平衡很难精确测量单个蚊子的重量，因此无法通过测量重量轻松量化单个膳食大小的变异性。仅建议女性明显在用餐时进行称重。<ol>
<li>冷麻醉蚊子通过移动他们的容器到4°C的冷室或把它放在冰上。</li>
		<li>称量组5女性从未提供一组（即蚊子从来没有提供一顿饭），并计算他们的平均体重作为估计的"喂养前"重量。未受蚊子的平均重量取决于基因型、性别和饲养条件。未受雌性 Ae. aegypti 蚊子饲养， 与 广告利比图姆 接触蔗糖通常每个重约 2 毫克。</li>
		<li>从实验队列（即提供膳食的蚊子），根据眼睛7可观察到的腹部分量，将雌性分成"喂"和"不喂"的桩。将每个"喂"和"不喂"的堆分别分成5组蚊子进行称量。每组5人中的蚊子应来自同一实验组，用于进行组重量测量。计算实验组每个"喂养"和"未喂养"堆中每个女性的平均体重。</li>
	</ol></li>
	<li>荧光测量，用于终点分析7,27,34 注意：要从不再需要进一步活体实验的单个蚊子那里获得精确的膳食大小测量，请在进食后立即将含有0.002%荧光素的1mL食物储存在-20°C。实验可以在这里暂停。此方法概述在 图2.<ol><li>要生成参考标准曲线，则准备对提供给实验组蚊子的含有 0.002% 荧光素的同一餐进行连续稀释。总共将有 8 个标准曲线解决方案。在每种解决方案中，含有 0.002% 荧光素的最终膳食量为 5、2.5、1.25、0.625、0.3125、 0.15625， 0.078125， 或 0 μL， 每个将在 1x 磷酸盐缓冲盐水 （PBS） 总体积 100 μL （例如， 5 μL 的膳食含有 0.002% 荧光素在 95 μL 的 1x PBS）.</li>
		<li>要使标准曲线的第一个解决方案，添加 50 μL 的膳食含有 0.002% 的荧光素到 950 μL 的 1x PBS 和漩涡彻底（最终体积：5 μL 的膳食含有 0.002% 的荧光素在 95μL 的 1x PBS）。要制作其余标准曲线解决方案，请从上一个管子中取出 500μL 并将其添加到包含 500μL 1 倍 PBS 的新管中，为每一步执行 2 倍稀释。在准备下一次 2 倍稀释之前， 漩涡很好。</li>
		<li>要准备用于生成参考标准曲线的油井，在 96 井 PCR 板的第一列中，每个标准曲线解决方案的移液器 100 μL 进入每个 8 口油井。在板的第一列相同的 8 口井中添加 1 个未受控制的蚊子。在板的第二列中重复以进行复制测量。 注：如果实验组提供不同的膳食类型，则必须为每种膳食类型准备一个单独的参考标准曲线。</li>
		<li>在未对照组和实验组的每个剩余井中添加 100 μL 的 1x PBS。如果组织要在随后的步骤中使用珠磨同质化剂或旋涡中断，则在每口井中添加一个 3 mm 的玻利西酸固体玻璃珠。</li>
		<li>作为负控制，在板的后 2 列中，在每口井中添加 1 只未清除的蚊子。该组测量的荧光设置基线截止，以解释组织自荧光，并将用于确定实验组中的蚊子是否在膳食中喂养。</li>
		<li>在提供膳食的实验组的剩余油井中，每口井增加1只蚊子。</li>
		<li>小心密封板，通过手动研磨破坏组织。腹部应彻底同质化以释放膳食。破坏组织的方法包括使用珠磨机同质化剂，具有3毫米的波罗西酸固体玻璃珠（30赫兹30秒），涡流搅拌机与3毫米波罗西酸盐固体玻璃珠，或无珠子的刺磨机。</li>
		<li>以 2000 rpm 的速度将盘子离心 1-2 分钟，以收集碎液。</li>
		<li>准备一个黑色的 96 井板，每口井有 180μL 的 1x PBS。</li>
		<li>将 20 μL 的利酸盐转移到每口井，并混合 180μL 的 1x PBS 并混合。如果可用，请在此步骤中使用多通道移液器，以提高速度和更好的一致性。</li>
		<li>使用 485/520 激发/排放通道上的板式读取器测量每口井的荧光强度。通过根据相应的荧光强度测量绘制已知的膳食量，生成参考标准曲线。</li>
		<li>使用生成的参考标准曲线，推断每个实验组蚊子摄入的膳食量。从每个实验组个体的荧光强度读数中减去未受蚊子负对照组的平均荧光强度读数，以校正基线组织自荧光。</li>
	</ol></li>
</ol>

<ol>
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作者没有什么可透露的。

对于许多实验室应用，人工膜喂食器通过让研究人员能够直接操纵餐食内容，与活体宿主相比具有明显的好处。虽然有多种方法可用于人工膜喂养，但此处描述的方法在灵活性、成本和吞吐量方面具有优势。与其他商业膜喂食器相比，Glytube检测需要少量的膳食量，通过最小化所需的总体积7，35，使其成为昂贵的试剂（包括药物或病原体）的有效输送机制。由于无蛋白盐水和人工血餐都促进活力，化合物或病原体可以添加到任何一餐作为高通量和非侵入性替代注射。此外，Glytube 的每个组件都很容易被洗涤、更换或放大，以提供和量化多种膳食类型，而不会交叉污染喂养设备。
为了量化蚊子的膳食量，荧光法比在喂食前后称量蚊子更精确的膳食大小量化。应当指出，这种方法是一种终点检测。相比之下，称量使蚊子能够存活下来进行进一步的实验。通过使用板式读取器，基于荧光的方法可以轻松扩展，以高通量量化数百名女性食用的膳食。
为了达到高喂养率，必须存在足够的宿主提示组合，以激活女性寻宿主行为，并吸引女性到喂食器。如果蚊子没有挤在 Glytube 下面，膳食可能无法适当加热，或者二氧化碳 的输送可能不够。在膜表面添加人类气味可可靠地增加人造膜的吸引力。如果在 Glytube 下观察到蚊子，但无法进食，则膳食成分可能有错。如果膳食本身不温暖，血液太旧，或者如果膳食中的添加剂本质上是反常的，或引起不良的化学反应，女性可能无法进食。额外的ATP还可以可靠地提高喂养率，并且可以在所提供的每个食谱中扩展到2mM的最终浓度。如果准胶片没有拉紧格莱图帽，女性可能无法进食：护身膜应均匀透明，不应扣扣，因为这样可以防止女性能够有效地用她的风格刺穿准胶片。如果餐食通过 Glytube 泄漏到网状物上，则准胶片可能在拉伸过程中撕裂，应更换。
改变膳食成分还可以使研究人员能够操纵从中餐中清除膳食所需的时间长度以及随后的寻宿行为。这里提供的膳食需要24-36小时消化7 类似于动物源性血液。吃完这些饭菜后，雌性会在消化时间窗口内抑制寻找宿主。由于盐水餐缺乏蛋白质，女性在餐后返回寻求宿主。如果更快的回报是可取的，研究人员可以选择替代"快速清除"盐水餐，排泄在大约6小时27。虽然这里呈现的盐粉成分与人工血餐直接比较结果，但"快速清除"膳食更符合脊椎动物血液中的生理盐含量。
此处描述的方法有局限性，在选择最适合研究人员实验目标的测定之前应考虑这些限制。所述的荧光素测量不允许蚊子再次用于额外的实验。但是，可以使用荧光素检测在膳食大小量化之前进行体重测量。如果给定膳食的多个试验中体重和膳食大小是一致的，则重量可以用作未来实验的代理。此外，该议定书没有区分寻求宿主和喂血行为的缺陷：发现膜喂食器有损伤的蚊子会降低进食率和/或膳食大小。通过添加摄像头来记录整个测定过程中的行为，研究人员可以确定雌性是否找不到Glytube，或者他们是否找到了Glytube，但不会进食。
此处描述的检测可以适应探索与蚊子喂养行为相关的许多未决问题。例如，可以通过改变成分蛋白的比例或人工血餐中蛋白质总浓度来探讨特定血液蛋白的贡献。为了评估多个喂养事件的膳食大小，可以添加具有明显荧光光谱的染料，以区分膳食与独特来源37。该协议也可以修改，以单独刺激内口部分检测血液和用于摄入（即风格），和化疗附属物接触皮肤（即阴唇，腿）作为蚊子土地开始血液喂养36。例如，如果将配体直接添加到膳食中，它们不会接触阴唇和腿部，因为膜仅通过样式穿孔。如果配体被添加到副膜的外表面，他们仍然与膳食分离，并可能接触的实验室和腿36。最后，对血液喂养行为的详细动能没有很好的理解，这里提出的方法可以修改为将高分辨率跟踪与机器学习工具相结合，以提取运动、姿势和喂食动力学的行为读数38。
该协议旨在用户友好且具有成本效益，能够为使用药理学和基因操作来研究蚊子血液喂养和血液喂养后行为的研究人员提供服务。

我们提出了一个协议，使用人工膜喂食器向 伊 蚊提供改性血液膳食，并精确测量每只蚊子的膳食量。此协议可以灵活地调整，以改变膳食内容或比较不同实验组蚊子消耗的膳食量。
艾吉普提蚊子传播病原体，导致黄热病、登革热、基孔肯雅病和寨卡1、2、3、4、5等疾病，威胁全球健康。艾吉普蒂女性是义务献血者：他们必须消耗脊椎动物的血液，以获得卵子发育所需的蛋白质，每个离合器的鸡蛋需要从至少一个主机6，7，8全血餐。雌性蚊子首先咬她的主人，用她的风格刺穿皮肤和注射唾液，其中含有化合物，触发宿主的免疫反应9。然后，她通过抽血通过她的风格到她的中口水喂养。在食用受感染宿主的血液餐时，她可能会摄入血液传播的病原体6、8，然后从蚊子的中游转移到唾液腺10。以这种方式感染的雌性蚊子在叮咬随后的宿主11、12时，会通过注射病原体和唾液来传播疾病。了解和量化供血行为的机制是控制蚊媒疾病传播的关键步骤。
许多用于蚊子饲养和实验的实验室协议使用活体动物，包括老鼠、豚鼠或人类作为血源13、14、15、16。活体动物的使用对人员培训、动物住房和护理以及遵守机构动物护理和使用委员会（IACUC）的政策提出了道德问题和复杂的要求。它还限制了可以输送到蚊子的化合物的类型，这限制了可以进行的研究17。
人工供血设备通常使用膜系统来模拟宿主皮肤，是研究血液喂养行为的有用工具，可以规避维持活体宿主的需要。全血可以从一些供应商购买，并喂蚊子使用加热，水衣人造膜喂食器或类似的设备18，19。在此协议中，我们演示了使用称为"Glytubes"的小型一次性膜馈线器。这种膜喂食器以前由科斯塔-达-西尔瓦等人（2013年）20日出版，可以很容易地从标准的实验室设备组装，使其非常适合向适量的蚊子提供血液膳食，并直接扩大规模，以测试更大的群体或多种膳食配方。Glytube 是一种廉价和高效的替代其他商业人工喂食器，它可能需要更大的膳食量，更适合批量喂养大群蚊子的单餐配方21。
该协议包括两个部分：准备/提供人工餐食和量化消费。在第一节中，Glytubes 被用作提供操纵饮食的有效手段。全血可以用完全人造的膳食代替，以比较血液替代品代替血液餐的效果。一个食谱改编自科根（1990）22在这里提出，虽然多个人造膳食配方已经开发23，24。此外，与注射相比，喂养是一种侵入性更小、更不费力的方法来引入药理化合物。由于每餐所需的总容量较低（1-2 mL），此协议提供了一个有吸引力的递送方法，以减少昂贵的试剂量。Ae. aegypti女性容易食用无蛋白质的盐水溶液与腺苷 5 +三磷酸酯 （ATP）25，26，它提供了一个基线，用于测量单膳食成分的影响.例如，Ae.aegypti的神经肽 Y 样受体 7 （NPYLR7） 已知在富含蛋白质的血液餐后会调解宿主求抑制，当 NPYLR7 激动剂添加到无蛋白质盐水餐中时，雌性蚊子表现出类似于那些消耗了全血的雌性蚊子寻求宿主的抑制。
在第二节中，介绍了量化雌性蚊子每餐消费量的步骤。这种测定基于荧光，捕获喂养状态的分辨率高于女性根据腹部分化的视觉评估被归类为"喂养"或"未喂养"的方法。通过在进食前将荧光素添加到膳食中，个人摄入的膳食量可以通过将每只蚊子同质化在 96 口井盘中并测量荧光强度作为读取来量化。这种检测可以测量喂养活力的差异，以响应诸如膳食成分或蚊子的遗传背景等变量。精确量化对于中餐规模至关重要，例如，当女性获得含有喂养威慑力的次优膳食时，或当她们食用可变大小为27的蔗糖餐时。如果餐量大小量化后需要喂养蚊子进行后续行为检测，则可以通过分组称量麻醉女性并估计每个人平均增加的质量来计算膳食大小。虽然重量不如荧光素标记精确，但仍提供膳食体积的汇总估计值，并允许检查膳食对下游过程的影响，如粪便或随后的宿主吸引力。虽然血餐的大小是可变的，并可能受到无数因素的影响11，28，29，摄入膳食大小测量与这里描述的方法是一致的以前的量化7，30，31。

<table>
	<tbody>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mm diameter borosilicate solid-glass bead</td>
			<td>MilliporeSigma</td>
			<td>Z143928</td>
			<td>For use for bead mill homogenizer; not required if using pellet pestle grinder</td>
		</tr>
		<tr>
			<td>32 oz. high-density polyethylene (HDPE) plastic cup</td>
			<td>VWR</td>
			<td>89009-668</td>
			<td>Example mosquito container used for feeding assays shown; alternate options can be used</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well black polystyrene plate</td>
			<td>ThermoFisher</td>
			<td>12-566-09</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well PCR plate sealing film</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>96-well PCR plates</td>
			<td>Bio-Rad</td>
			<td>HSP9621</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate (ATP) disodium salt hydrate</td>
			<td>MilliporeSigma</td>
			<td>A6419</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin (human serum)</td>
			<td>MilliporeSigma</td>
			<td>A9511</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher Scientific</td>
			<td>01-213</td>
			<td>Alternate options can be used to block light entering fluorescein container</td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Fisher Scientific</td>
			<td>01-911</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Bead mill homogenizer</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>Not required if using pellet pestle grinder</td>
		</tr>
		<tr>
			<td>Cotton ball</td>
			<td>Fisher Scientific</td>
			<td>22456880</td>
			<td>For nectar-feeding; alternate options can be used</td>
		</tr>
		<tr>
			<td>Defibrinated sheep blood</td>
			<td>Hemostat Laboratories</td>
			<td>DSB100</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Drosophila CO2 fly pad</td>
			<td>Tritech Research</td>
			<td>MINJ-DROS-FP</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Fluorescein</td>
			<td>MilliporeSigma</td>
			<td>F6377</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence plate-reader</td>
			<td>ThermoFisher</td>
			<td>VL0000D0</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Gamma-globulin (human blood)</td>
			<td>MilliporeSigma</td>
			<td>H7379</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>MilliporeSigma</td>
			<td>G7893</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemoglobin (human)</td>
			<td>MilliporeSigma</td>
			<td>G4386</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory wrapping film - parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>Fisher Scientific</td>
			<td>90-691</td>
			<td>Alternate magnetic stirrers can be used</td>
		</tr>
		<tr>
			<td>Microcentrifuge for 96-well plate</td>
			<td>VWR</td>
			<td>80094-180</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes</td>
			<td>MilliporeSigma</td>
			<td>2236412</td>
			<td>Alternate options can be used</td>
		</tr>
		<tr>
			<td>Pellet pestle grinder</td>
			<td>VWR</td>
			<td>KT749521-1500</td>
			<td>Not required if using bead mill homogenizer</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BW17-516F</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td>Alternate options can be used, such as a lathe for better consistency of cutting</td>
		</tr>
		<tr>
			<td>Rubber bands</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone tubing</td>
			<td>McMaster Carr</td>
			<td></td>
			<td>Needed if using a fly pad for CO2 delivery</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Fisher Scientific</td>
			<td>S233</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>MilliporeSigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir bars</td>
			<td>Fisher Scientific</td>
			<td>14-512</td>
			<td>Alternate magnetic stir bars can be used</td>
		</tr>
		<tr>
			<td>Translucent polypropylene storage box with removable lid</td>
			<td></td>
			<td></td>
			<td>Example box used for feeding assays shown</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td>Alternate heating device may be used</td>
		</tr>
		<tr>
			<td>White 0.8 mm polyester mosquito netting</td>
			<td>American Home &#38; Habit Inc.</td>
			<td>F03A-PONO-MOSQ-M008-WT</td>
			<td>Alternate options can be used</td>
		</tr>
	</tbody>
</table>

feeding and quantifying animal-derived blood and artificial meals in aedes aegypti mosquitoes

某些蚊子物种的女性可以传播疾病，同时咬脊椎动物宿主，以获得卵子发育所需的富含蛋白质的血液膳食。在实验室里，研究人员可以通过膜喂食器向蚊子提供动物源性血液和人造血液餐，从而能够操纵膳食成分。在这里，我们 介绍的方法， 喂血和人工血液餐伊蚊和量化个别女性的消费量。
有针对性的喂养和定量人工/血液膳食具有广泛的用途，包括测试膳食成分对蚊子行为和生理的影响，不注射就提供药理化合物，以及用特定病原体感染蚊子。在进食前将荧光素染料添加到膳食中，可进行后续膳食尺寸的量化。蚊子消耗的膳食量可以测量体重，如果女性以后要用于行为实验，或者通过在96口井板中使个体雌性同质化，并使用板读器作为终点检测测量荧光水平。膳食大小量化可用于确定更改膳食成分是否会改变摄入的膳食量，或者蚊子菌株之间的膳食消耗是否不同。精确的膳食大小量化对于下游检测也至关重要，例如测量对宿主吸引力或丰盛度的影响。这里介绍的方法可以进一步适应跟踪膳食消化在几天内，或包括多个可区分的标记添加到不同的膳食（如花蜜和血液），以量化每餐由一只蚊子的消费。
这些方法使研究人员能够单枪匹马地进行高通量测量，以比较数百只蚊子的膳食量。因此，这些工具对于蚊子研究人员群体回答各种生物问题将大有用处。

图 1提供了组装 Glytube 的示意图，而图 2显示了使用此处描述的基于荧光的检测来测量膳食大小的实验设计概述。图 3提供了血液喂养实验中具有代表性的荧光素膳食尺寸测量结果。图 4、图 5和图 6显示了可使用本协议解决的生物问题的样本。该协议的应用范围很广，包括改变血粉成分、喂养药理化合物、精确量化次优血餐或小型花蜜餐，以及比较蚊子基因型的喂养行为。
为了生成膳食量计算的标准曲线，荧光读数从指定的参考井中绘制，每个油井都含有未受蚊虫和已知卷，含 0.002% 荧光素（图 3A）。剩余水井的荧光读数与此标准曲线进行比较，以量化每只蚊子消耗的膳食量 （μL） （图 3B）。要验证此检测中的基线读数，应确认未受负对照组的蚊子未分配消耗的 μL 正值（图 3B，左图）。虽然实验组的所有女性都得到了血餐，但有些蚊子喂食（图3B，中间），有些没有（图3B，右图）。这一结果表明，可以从本协议中获取两种类型的数据：1） 以给定膳食为食的女性总数的百分比，以及 2） 以给定膳食为食的女性摄入的体积。
此协议可用于提供和量化含有各种蛋白质成分的膳食。 图 4A，B 显示使用添加荧光素的膳食收集的数据。喂养的蚊子比例和它们摄入的膳食量分别根据荧光读数计算。这些读数非常敏感，允许精确量化μL，但有蚊子不能用于未来活体实验的局限性。 图4C，D 显示了从对蚊子的独立实验中收集的数据，这些蚊子在没有荧光素的膳食后被评分为喂养或没有眼睛。膳食大小计算为5组蚊子的平均体重/雌性。虽然这些重量测量比荧光测量更不敏感，但它们允许女性被恢复并用于进一步的活体实验。喂养的蚊子比例可能因实验日而异，如 图4A 和 图4C所反映的那样。
图 5 显示了含有调节蚊子宿主寻找行为的药物的膳食消耗量。在这些实验中，女性获得血液、盐水+ATP或盐水+ATP餐食，其中100μM为人类NPY Y2受体激动剂TM30338。这种药物通过激活 类似 Ae. aegypti NPY 的受体 7 来改变寻宿主行为。测量膳食大小对于解释实验以评估这种药物对血液喂养后行为的影响至关重要，因为它允许研究人员计算每个女性消耗的剂量。
在以前的例子中，女性要么喂血，要么用血代替，所有这些都导致3-5μL餐（图3，图4，图5）。这种基于荧光的检测方法还可用于测量从平均组重量测量中无法准确辨别的更小和/或更可变的膳食大小。在图6中，同样的荧光量化方案被用来测量花蜜喂养行为，用Glytube交换一个棉球饱和10%的蔗糖含有0.002%的荧光素。花蜜糖不能在Glytube检测中呈现，因为女性无法检测花蜜糖的存在与风格，不启动喂养27。这些数据使研究人员能够确定糖膳食总是小于血餐，这与以前的工作34（图6）一致。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61835/61835fig1v2.png"> 图1：设置用于向蚊子喂食的Glytube方法。（A） 用于向蚊子喂血和其他膳食的解构胶管的示意图。（B） 格利图布的示意图呈现在一个带网状盖的蚊子容器上。雌性蚊子可以穿透网盖喂食。（C） 格利图布（上图）和雌性伊蚊在喂食前、喂食期间和喂食后（从下到右）的照片。蚊子通过覆盖容器的网眼穿透，进入膜馈线。（D） 照片显示雌性Ae. aegypti蚊子的外观，这些蚊子没有（左），并且已经注入了人工血餐（右，顶部）或盐水 + ATP 餐（右，下）。格利图布方法之前发表在科斯塔-达-西尔瓦等人（2013）20。照片在 （C） 和（D） 由亚历克斯 · 威尔德提供。<a href="https://www.jove.com/files/ftp_upload/61835/61835fig1v2large.png" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61835/61835fig2v4.png"> 图2：胶管供血协议后如何量化膳食大小的示意图。（A） 蚊子提供荧光素（顶部，实验组）或无餐（底部，无负对照组）的膳食。（B） 终止喂食实验后，个别蚊子会加入96井盘内。（C） 标准曲线是使用已知含有0.002%荧光素的膳食量生成的。（D） 蚊子是同质化的，以释放任何消耗的荧光素，每个井的荧光水平使用板读取器进行量化。这种荧光量化方法从利施等人（2013）34中修改而成。<a href="https://www.jove.com/files/ftp_upload/61835/61835fig2v4large.png" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61835/61835fig3v2.png"> 图3：用基于荧光素的量化进行胶管供血实验。（A） 从未受控制组的蚊子被添加到已知数量的膳食中，含有0.002%荧光素（y轴尺度=任意单位）的井中获得的标准曲线测量结果。（B） 餐量使用未对照组（左、黑、n =40）、以血液为食的实验组（中、红色、n = 37）和不以血液为食的实验组（右、红、n=23）的女性计算。每个点代表来自女性个体的测量结果。数据显示为带范围的中位数。字母表示统计学上不同的组，克鲁斯卡尔-瓦利斯测试与邓恩的多重比较，p<0.01。这些数据发表在Jové等人（2020）27。<a href="https://www.jove.com/files/ftp_upload/61835/61835fig3v5large.png" target="_blank">请点击这里查看此数字的较大版本。</a>

<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61835/61835fig5v2.png"> 图5：用药理化合物量化膳食。女性食用相同大小的羊血（红色）、盐水+ATP（水）和盐水+ATP+100 μM剂量的人类NPY Y2受体激动剂TM30338（深蓝色）。使用荧光读数计算的膳食量。每个点代表来自女性个体的测量结果。数据显示为带范围的中位数，n = 12。字母表示统计学上不同的组，克鲁斯卡尔-瓦利斯测试与邓恩的多重比较，p<0.05。 <a href="https://www.jove.com/files/ftp_upload/61835/61835fig5v5large.png" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61835/61835fig6v2.png"> 图6：小花蜜餐的量化。（A） 花蜜喂养检测的示意图。（B） 在花蜜喂养检测中，使用野生型雌性荧光读数计算的膳食量提供水（蓝色、n = 36）或10%蔗糖（绿色，n = 53），每餐均带有0.002%的荧光素。每个点代表来自女性个体的测量结果。数据显示为带范围的中位数。字母表示统计学上不同的组，曼恩-惠特尼测试，p<0.05。这些数据发表在Jové等人（2020）27。<a href="https://www.jove.com/files/ftp_upload/61835/61835fig6v5large.png" target="_blank">请点击这里查看此数字的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="4">人造血餐</td>
		</tr>
<tr>
<td></td>
			<td>库存解决方案的集中（毫克/mL）</td>
			<td>膳食中库存解决方案的体积 （μL/mL）</td>
			<td>最终膳食浓度（毫克/mL）</td>
		</tr>
<tr>
<td colspan="4">蛋白质成分*</td>
		</tr>
<tr>
<td>γ-球蛋白</td>
			<td>50</td>
			<td>300</td>
			<td>15</td>
		</tr>
<tr>
<td>血红蛋白</td>
			<td>35</td>
			<td>230</td>
			<td>8</td>
		</tr>
<tr>
<td>白 蛋白</td>
			<td>300</td>
			<td>340</td>
			<td>102</td>
		</tr>
<tr>
<td>蛋白质总量</td>
			<td>-</td>
			<td>-</td>
			<td>125</td>
		</tr>
<tr>
<td colspan="4">非蛋白质组件</td>
		</tr>
<tr>
<td></td>
			<td>库存解决方案的集中 （mM）</td>
			<td>膳食中库存解决方案的体积 （μL/mL）</td>
			<td>最终膳食浓度 （mM）</td>
		</tr>
<tr>
<td>Nacl</td>
			<td>以γ球蛋白股票</td>
			<td>-</td>
			<td>5-10</td>
		</tr>
<tr>
<td>纳科3
</td>
			<td>以γ球蛋白股票</td>
			<td>-</td>
			<td>120</td>
		</tr>
<tr>
<td>Atp</td>
			<td>200</td>
			<td>5</td>
			<td>1</td>
		</tr>
<tr>
<td>水</td>
			<td>-</td>
			<td>125</td>
			<td>-</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td colspan="4">*蛋白质成分在双蒸馏水的库存溶液中制备，γ球蛋白除外，这些蛋白溶解在 400 mM NaHCO3 中，包括可变量的 NaCl （2-4%）在产品中。</td>
		</tr>
</tbody></table>表1：制备人工血餐的食谱（改编自科根（1990）22）。人造血液由人类血液中经常发现的蛋白质和非蛋白质成分组成，并提供改变这些成分比例的选择。蚊子可以产生鸡蛋后，喂养人工血液7，22。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="4">盐粉</td>
		</tr>
<tr>
<td>组件</td>
			<td>库存解决方案的集中 （mM）</td>
			<td>膳食中库存解决方案的体积 （μL/mL）</td>
			<td>最终膳食浓度 （mM）</td>
		</tr>
<tr>
<td>Nacl</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
<tr>
<td>纳科3
</td>
			<td>400</td>
			<td>300</td>
			<td>120</td>
		</tr>
<tr>
<td>Atp</td>
			<td>200</td>
			<td>5</td>
			<td>1</td>
		</tr>
<tr>
<td>水</td>
			<td>-</td>
			<td>695</td>
			<td>-</td>
		</tr>
</tbody></table>表2：与ATP的盐水餐食谱（改编自杜瓦尔等人（2019）7）。无蛋白盐水餐可用于向蚊子提供感兴趣的化合物，同时仍模仿血液喂养后发生的腹部分化，但不会触发蛋白质摄入时发生的卵子发育。

Watch this Scientific Journal Video about Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes at JoVE.com

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

该议定书的目标是通过人工膜喂食器向 伊 蚊提供动物源和人工血液膳食，并精确量化摄入的膳食量。

在 伊 蚊中喂养和量化动物衍生的血液和人工膳食

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

feeding-quantifying-animal-derived-blood-artificial-meals-aedes

Research

JoVE Journal

Immunology and Infection

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

7.6K 次观看.  The Rockefeller University. 该议定书的目标是通过人工膜喂食器向 伊 蚊提供动物源和人工血液膳食，并精确量化摄入的膳食量。

视频: Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

荧<em在原位</em&gt;蚊虫的有丝分裂染色体上的杂交

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

在 伊 蚊中喂养和量化动物衍生的血液和人工膳食

在伊蚊中喂养和量化动物衍生的血液和人工膳食