N.G.是法国"法国重建基金会"和菲利普基金会的奖学金获得者;R.S. 由露西尔·帕卡德儿童健康基金会和斯坦福NIH/NCRR CTSA奖号码UL1 RR025744支持;P.S. 得到马克斯·卡德基金会和奥地利科学院的马克斯·卡德研究金和奥地利科学基金（FWF）施罗德研究金的支持：J3399-B21：S.J.G. 承认来自国家卫生研究院的支持，资助 U19 AI104209、NS 080062 和加州大学烟草相关疾病研究计划;L.L.R. 承认关节炎国家研究基金会 （ANRF） 和国家卫生研究院资助 K99AI110645。

所有动物护理和实验都是按照国家卫生研究院的指导方针和斯坦福大学动物护理和使用机构委员会的具体批准进行的。
1. 骨髓衍生培养乳腺细胞（BMCMCs）的生成和特征。
注意：捐赠者BMCMC应该来自与接受MC缺乏小鼠具有相同遗传背景的骨髓细胞。雄性供体BMCMC不适合雌性小鼠的移植。女性衍生的捐赠者BMCMC将成功地融入男性和女性接受者。
<ol><li>骨髓提取。<ol>
<li>将无菌磷酸盐缓冲盐水 （PBS） 倒入 6 个井培养板（每只小鼠 1 口井），并放在冰上。</li>
		<li>将解剖仪器浸泡在70%的乙醇中进行灭菌。</li>
		<li>通过吸入二氧化碳（CO2）然后进行颈椎脱位，然后用70%的乙醇在操作现场喷洒小鼠安乐死。</li>
		<li>使用无菌解剖仪器提取股骨、头骨和腓骨，而无需切割任何骨表皮。</li>
		<li>在用钳子固定骨骼时，用无菌剪刀（或手术刀片）刮掉骨头上的所有组织（并丢弃腓骨）。将骨骼放在 PBS 上，直到收集所有骨骼。</li>
		<li>在无菌组织培养罩中执行所有剩余步骤。使用无菌仪器，用钳子固定骨骼，并切断两个表皮，以暴露髓腔。</li>
		<li>使用 3 毫升注射器和 30 G 针头，以及冷冲洗介质（杜尔贝科改性鹰介质 [DMEM] 补充 10% 胎儿小腿血清 [FCS， 热灭活]， 2 mM L-谷氨酰胺， 1% 抗生素抗菌溶液， 50 μM β-mercaptoethanol）， 将红骨髓冲入培养皿中。</li>
		<li>使用相同的注射器通过反复的温和吸气和弹出来分离冲洗的骨髓细胞簇。</li>
		<li>将每只小鼠的股骨和二头肌骨髓池放入一个15毫升的离心机管中。用冷冲洗介质填充管子，在 4°C 下以 400 x g 5 分钟的速度清洗骨髓细胞和离心机 5 分钟。</li>
	</ol></li>
	<li>宝马文化。<ol>
<li>去除超自然，在10毫升培养介质中恢复盆栽骨髓细胞（DMEM补充10%胎儿小腿血清[FCS， 热灭活]，2米L-谷氨酰胺，1%抗生素抗菌溶液，50μM+-麦加托乙醇和20%WEHI-3细胞条件介质[作为IL-3的来源;或者使用重组小鼠IL-3在10 ng/ml]）。</li>
		<li>将细胞放在大小合适的组织培养瓶中（例如，T25代表1只小鼠，T75代表2只小鼠 等）。</li>
		<li>电镀细胞后1-2天，将介质和悬浮细胞（留下碎片和粘在烧瓶底部的粘附细胞）转移到新的烧瓶中，并添加新鲜培养介质（10毫升/鼠标）。</li>
		<li>在接下来的几周内，每3-4天喂一次细胞（每只小鼠添加10毫升培养介质）。保持2.5 x 105-1 x 106 细胞/毫升之间的细胞密度。每周将非粘附细胞转移到新的烧瓶中一次，直到培养瓶中不存在粘附细胞。 在用于体外 检测或移植到缺乏MC的小鼠之前，先测试细胞的成熟度（见下文）。 注：BMCMC的完全分化需要4-6周。</li>
	</ol></li>
	<li>评估 BMCMC 成熟度。<ol>
<li>
使用流细胞测量。
		<ol>
<li>在 5 毫升聚苯乙烯圆形底部管中使用冰冷 FACS 缓冲（PBS，0.5% FCS）清洗细胞（5 x10 4-5 x 10 5 细胞/条件）。离心机在400 x g 5分钟在4°C。 吸气超自然。</li>
			<li>在 FACS 缓冲区中稀释防鼠器 CD16/32（克隆 93 或 2.4G2）单克隆抗体 1：200 （2.5μg/ml）。在每个颗粒中加入 10 μl，然后通过短暂的漩涡来补充。在冰上孵化 5 分钟以阻止 Fc 绑定。 注意：保护样品免受所有剩余步骤的直接光。</li>
			<li>稀释植物素 （PE） 结合抗鼠标 Fc εRI α （克隆 MAR-1） 1：200 （1μg/ml） 和荧光素异硫氰酸酯 （FITC） 结合抗鼠标 KIT （CD117; 克隆 2B8） 1：200 （2.5μg/ml） （"染色溶液"）， 或在 FACS 缓冲器中分别标记的同型控制抗体 （"同型控制解决方案"）。</li>
			<li>将一半的阻塞细胞从步骤 1.3.1.2 分裂成额外的聚苯乙烯圆形底部管，并在一个管中加入 20 μl 的"染色溶液"，在另一个管中加入 20 μl 的"异型控制解决方案"。在冰上孵化30分钟。</li>
			<li>在 400 x g 下加入 3 毫升冰冷 FACS 缓冲器和离心机，在 4 °C 下 5 分钟。 丢弃超自然物，将颗粒重新喷入含有 1μg/ml 碘化物 （PI， 用于识别死细胞） 的 300μl FACS 缓冲器中。继续进行流动细胞学分析（见图2A）。</li>
		</ol>
</li>
		<li>使用托卢伊丁蓝色染色。<ol>
<li>在室温下，通过离心在 400 x g 下使用 PBS 清洗 1 x 105 个细胞 5 分钟，然后在 200μl 的 PBS 中吸气并重新吸附细胞。</li>
			<li>将细胞悬架转移到连接到显微镜幻灯片的预制细胞元件中，然后使用细胞中心燃料（40 x g 5 分钟）进行细胞离心处理。</li>
			<li>空气干燥滑动10分钟，用PAP笔在细胞周围画一个蜡圈。添加 0.1% 的托卢伊丁蓝色溶液以覆盖细胞。孵化1分钟。在自来水中清洗滑梯1分钟，然后空气干燥滑梯10分钟。</li>
			<li>使用安装介质覆盖滑动单元。继续进行光显微镜分析（见图2B）</li>
		</ol>
</li>
	</ol></li>
</ol>2. 用BMCMC对乳腺细胞缺乏小鼠进行移植。
<ol><li>通过皮肤内注射（即）注射使耳朵受包。 注意：对于将BMCMC引入MC缺陷小鼠的耳钉的采用性转移，我们建议对每只耳针进行两次注射，每个细胞1 x10 6个细胞（共2 x10 6个细胞）。许多研究者在多个模型中都使用过将BMCMC引入MC缺陷小鼠耳钉的皮内移植，包括被动性皮肤过敏（PCA）24，27模型、宿主对毒液28的防御模型和慢性过敏反应（CHS）29，30。<ol>
<li>在冷 DMEM 中以 4 x 107 BMCMC/ml（1 x 106 BMCMC/25 μl）计算和补充细胞。将 BMCMC 解决方案转移到装有 30 G 针的 1 毫升注射器中。保持冰上，直到注射。</li>
		<li>使用异氟素（2.5%v/v）麻醉4-6周大的MC缺陷小鼠。通过脚趾捏检查麻醉深度，如果指示，调整异氟素，并在整个过程中继续监测呼吸和脚趾捏反应。使用眼药膏与Q提示，以防止眼睛干燥。</li>
		<li>使用食指，在耳钉的后部面部产生垂直压力，以暴露和伸展心室面部。</li>
		<li>将两个 25μl 即 BMCMC 溶液注射到耳针腹腔表面的两个不同部位，第一次注射在耳朵中间，第二次注射到耳针的尖端。在 体内实验之前 ，等待4-6周后。 注意：根据我们的经验，到那时，耳针中央部的MC/mm2 数量通常与野生型小鼠的相应位置相似，而耳针周围部的MC/mm2 数量通常大大低于相应的野生型小鼠27，28。在设计此类 MC 移植小鼠的实验时，应牢记这一点（例如，对 MC 功能可能产生影响的制剂应注射到耳钉的中心部分，并且对此类治疗效果的分析也应侧重于该区域）。</li>
	</ol></li>
	<li>腹膜腔通过内皮注射（即）注射而屈服。 注：通过将BMCMC转移到缺乏MC的小鼠的腹腔中，需要给每只小鼠注射2×106 个细胞。许多小组都利用这种将BMCMC注射到缺乏MC的小鼠身上，研究腹膜MC在各种模型中的作用，包括宿主防御毒液31 或细菌败血症32，33的模型。<ol>
<li>在冷 DMEM 中，以 1 x 107 BMCMC/ml（2 x 106 BMCMC/200 μl）计算适当的细胞数量并重新发送。将 BMCMC 解决方案转移到装有 25 G 针的 1 毫升注射器中。保持冰上，直到注射。</li>
		<li>将 200μl BMCMCs 溶液注射到 4-6 周大的 MC 缺陷小鼠的腹腔中。在 体内实验之前 ，在i.p.移植后等待4-6周。</li>
	</ol></li>
	<li>静脉注射（即注射）。 注：通过注射到缺 MC 小鼠中，BMCMC 的采用转移需要每只小鼠注射 5 x 106个细胞。许多小组已经使用静脉注射BMCMC到MC缺乏小鼠，以研究MC在各种疾病模型中的作用，包括膀胱感染34模型，哮喘35，肺纤维化36，抗体介导关节炎37。<ol>
<li>在冷 DMEM 中计算适量的细胞，并以 2.5 x10 7 BMCMC/ml（5 x10 6 BMCMC/200μl）进行再喷销。将 BMCMC 解决方案转移到装有 30 G 针的 1 毫升注射器中。保持冰上，直到注射。</li>
		<li>使用异氟素（2.5%v/v）麻醉4-6周大的MC缺陷小鼠。通过脚趾捏检查麻醉深度，如果指示，调整异氟素，并在整个过程中继续监测呼吸和脚趾捏反应。使用眼药膏与Q提示，以防止眼睛干燥。</li>
		<li>将 200μl BMCMC 溶液注入 MC 缺陷小鼠的尾静脉（或者，或者，逆轨道静脉）中。在 体内实验之前 ，在i.v.移植后等待12周。</li>
	</ol></li>
</ol>3. 对受精的桅杆细胞缺乏小鼠的分析。
<ol><li>耳部移植分析。<ol>
<li>在实验结束时，通过CO2 吸入对小鼠实施安乐死，然后进行宫颈错位。</li>
		<li>隔离耳针，固定在10%（vol/vol）缓冲形式在4°C过夜。 将固定耳针嵌入石蜡中，切开 4 μm 部分耳针，并安装在玻璃滑梯上。</li>
		<li>在室温下用 0.1% 的托卢伊丁蓝色溶液染色滑梯 1 分钟。在自来水中清洗滑梯1分钟，然后在室温下将空气干燥滑梯洗10分钟。</li>
		<li>使用安装介质的盖滑动，然后使用光显微镜计算每个针头部分的镶有MC的数量。通过比较受 包小鼠 与野生小鼠耳皮中MC的百分比和分布来评估移植效率。</li>
	</ol></li>
	<li>腹腔移植分析。<ol>
<li>评估腹腔中的桅杆细胞数量。<ol>
<li>在实验结束时，通过CO2 吸入对小鼠实施安乐死，然后进行宫颈错位。小心地去除小鼠的腹腔皮肤，而不会破坏腹腔。</li>
			<li>使用配备 25 G 针的 5 毫升注射器在腹腔中注射 5 毫升冷或室温 PBS。使用冷PBS来降低激活腹膜细胞的风险（这在评估腹膜MC脱粒或腹膜熔岩液中某些MC衍生产品的水平时非常重要）。对腹部进行20秒的按摩，以收获腹膜细胞。</li>
			<li>使用配备 22 G 针的 5 毫升注射器慢慢吸气腹膜。记录吸气洗衣的体积（预计可恢复高达注入量的 80%）。</li>
			<li>将腹腔熔岩液转移到 5 毫升聚苯乙烯圆形底部管和离心机中，在 4°C 下 5 分钟 400 x g。 吸气超自然。在 400μl 冷 PBS 中恢复颗粒。</li>
			<li>使用血细胞计室计算腹膜细胞总数。使用细胞悬浮的一半进行流动细胞学分析（如步骤 1.3.1 中所述），以评估腹腔中受陷的 MCs 的百分比和标记表达。将腹腔细胞总数乘以MC的百分比，以获得腹腔MC的绝对数量。</li>
			<li>执行细胞集中处理与步骤 1.3.2.2 中描述的剩余细胞。空气干燥在室温下滑动10分钟，并用PAP笔在细胞周围画一个蜡圈。</li>
			<li>用未稀释的梅-格伦瓦尔德染色溶液覆盖细胞5分钟，然后在PBS中清洗5分钟，均在室温下。用Giemsa污渍覆盖细胞，用除离子水稀释1：20，在室温下孵育20分钟。在自来水中清洗滑梯2次1分钟，然后空气干燥滑梯。</li>
			<li>使用安装介质覆盖滑动单元，并使用光显微镜（通过计算至少 400 个总细胞）计算被灌装的 MCs 的百分比。通过比较被围困小鼠 与 野生小鼠腹腔厕所中的MC百分比来评估增殖效率。</li>
		</ol>
</li>
		<li>对机械窗中桅杆细胞的评估。<ol>
<li>继步骤 3.2.1 中描述的腹膜后，切开腹膜以暴露小鼠的肠道。将每个鼠标的 4 到 5 个感官窗口排列到幻灯片上。</li>
			<li>在室温下将滑梯固定在卡诺伊溶液（3：2：1卷/卷/伏乙醇、氯仿和冰川醋酸）中 1 小时。空气干燥滑动并移除肠道（固定的中位窗口将保持连接到滑梯上）。</li>
			<li>用 Csaba（阿尔西亚蓝/萨夫拉宁 O）染色溶液在室温下将准备时间染色 20 分钟。<ol>
<li>要制作 500 毫升 Csaba 污渍，通过混合 100 毫升 1 M 醋酸钠溶液和 120 毫升 1 M HCl 来制作 500 毫升醋酸缓冲液，用除离子水制作最多 500 毫升，并将 pH 调整为 1.42。然后，溶解 90 毫克 Safranin [识别红色中的'成熟 MCs]，1.8 g 阿尔西亚蓝色 [识别蓝色中的'不成熟的 MCs] 和 2.4 g 硫酸铵 NH4Fe （SO4）2到 500 毫升醋酸盐缓冲器。</li>
			</ol>
</li>
			<li>使用安装介质的盖滑动，然后使用光显微镜计算每个感官窗口的镶有MC的数量。通过比较被灌 装小鼠 与野生小鼠的间质窗口中MC的百分比和分布来评估增生效率。</li>
		</ol>
</li>
	</ol></li>
	<li>I.v. 恩格拉夫特分析。<ol>
<li>在实验结束时，通过二氧化碳 吸入和宫颈错位对小鼠实施安乐死，并收获感兴趣的组织（如 肺、皮肤或脾脏），并在4°C的10%（vol/vol）缓冲形式素中过夜固定。</li>
		<li>将固定组织嵌入石蜡中，切开 4 μm 部分，并安装在玻璃滑梯上。在室温下用 0.1% 的托卢伊丁蓝色溶液染色滑梯 1 分钟。在自来水中清洗滑梯1分钟，然后空气干燥滑梯10分钟。</li>
		<li>使用安装介质进行盖滑动，并使用光显微镜评估每个组织部分的受包MC的数量和分布。</li>
	</ol></li>
</ol>

作者没有什么可透露的。

在最初的描述38年之后的近30年，"桅杆细胞敲击"方法继续提供有价值的信息，说明MC在体内可以做什么或不能做什么。长期以来，人们一直认为MC的功能仅限于它们在过敏症中的作用。使用"桅杆细胞敲击"方法生成的数据改变了这一观点，通过提供证据，证明MC除了其他功能外，可以在宿主防御某些病原体4，39或毒气28，31方面发挥关键作用，甚至可以抑?...

乳腺细胞（MCs）是由多能骨髓祖先1-3产生的造血细胞。骨髓外泄后，MCs祖先迁移到各种组织，在当地生长因子1-3的影响下发育成成熟的MC。组织驻地MC战略性地位于主机环境界面，如皮肤、气道和胃肠道，它们作为抵御外部侮辱的第一道防线。MCs 通常根据其"基线"表型特征及其解剖位置进行子分类。在小鼠中，有两种类型的MC被描述："结缔组织型"MC（CTMC）和粘体MC（MMCs）1-3，7，8。CTMC通常位于静脉注射器和神经纤维附近，并居住在血清腔中，而MMC在肠道和呼吸粘膜1-3中占据腹腔内位置。
研究9-13年MC的生物功能，已应用多种方法。许多小组都专注于使用细胞系的体外方法（如人类MC线HMC114或LAD215，16）、体外衍生MC（如人类外周血源性MC17，或小鼠骨髓衍生） 培养的MC [BMCMCs]18，胎儿皮肤衍生MC [FSCMCs]19和腹腔细胞衍生MC [PCMC]20）或来自不同解剖站点的前体内分离的MC。所有这些模型被广泛用于研究MC生物学的分子细节，例如MC激活中涉及的信号通路。然而，MCs生物学的一个重要方面是，它们的表型和功能特征（如细胞质颗粒蛋白酶含量或对不同刺激的反应）可以通过解剖位置和微环境2，7进行调节。由于在体内遇到的这些因素的确切混合物可能很难在体外繁殖，我们赞成使用体内方法来深入了解MC功能9。
存在一些具有遗传 MC 缺陷的小鼠菌株，例如广泛使用的 WBB6F1-套件W/W-v或 C57BL/6-套件W-sh/W-sh小鼠。这些小鼠缺乏KIT（CD117）的表达和/或活性，KIT是主要MC生长因子干细胞因子（SCF）21，22的受体。因此， 这些小鼠有一个深刻的MC缺陷，但也有额外的表型异常有关他们的c-套件突变（在WBB6F1-套件W/W-v小鼠）或大染色体反转的影响，导致减少c-套件表达（在C57BL/6-套件W-sh/W-sh小鼠）9，10，12，23。最近，一些具有c-套件独立构成MC缺乏症的小鼠株被报告为24-26。所有这些小鼠和一些额外的新型诱导MC缺陷小鼠最近已详细审查9，10，13。
在这里，我们描述了小鼠骨髓衍生培养MC（BMCMCs）的生成方法，它们被收养转移到缺乏MC的老鼠，以及不同解剖部位对收养转移的MC的数量和分布的分析。这种所谓的 "桅杆细胞敲击" 方法可用于评估MC和MC衍生产品 在体内的功能。我们根据近年来开发的替代方法讨论了这种方法的优点和局限性。

<table border="0" cellpadding="0" cellspacing="0">
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		<tr>
			<td>1% Antibiotic-Antimycotic Solution</td>
			<td>Corning cellgro</td>
			<td>30-004-Cl</td>
			<td></td>
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		<tr>
			<td>3 ml Syringe</td>
			<td>Falcon</td>
			<td>309656</td>
			<td></td>
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			<td>35 mm x 10 mm Dish</td>
			<td>Corning cellgro</td>
			<td>430588</td>
			<td></td>
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		<tr>
			<td>5 ml Polystyrene Round Bottom Tube</td>
			<td>Falcon</td>
			<td>352058</td>
			<td></td>
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			<td>Acetic Acid Glacial</td>
			<td>Fisher Scientific</td>
			<td>A35-500</td>
			<td></td>
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		<tr>
			<td>Alcian Blue 8GX</td>
			<td>Rowley Biochemical Danver</td>
			<td>33864-99-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra 6R Centrifuge</td>
			<td>Beckman</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/32 (clone 93) Purified</td>
			<td>eBioscience</td>
			<td>14-0161-81</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 ml TB Syringe</td>
			<td>BD Syringe</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 22G x1 (0.7 mm x 25 mm) Needles</td>
			<td>BD Precision Glide Needle</td>
			<td>205155</td>
			<td></td>
		</tr>
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			<td>BD 25G 5/8 Needles</td>
			<td>BD Syringe</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 30G x1/2 Needles</td>
			<td>BD Precision Glide</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue MAX Jr, 15 ml Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytoseal 60 Mounting Medium</td>
			<td>Richard-Allan Scientific</td>
			<td>8310-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin3</td>
			<td>Shandon</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>DakoCytomation pen</td>
			<td>Dako</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle Medium (DMEM) 1x</td>
			<td>Corning cellgro</td>
			<td>15-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E 7023-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Heat Inactivated</td>
			<td>Sigma Aldrich</td>
			<td>F4135-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated IgG2b K Rat Isotype Control</td>
			<td>eBioscience</td>
			<td>14-4031-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein Isotiocyanate (FITC) Conjugated Anti-mouse KIT (CD117; clone 2B8)</td>
			<td>eBioscience</td>
			<td>11-1171-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>F79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Giemsa Stain Modified</td>
			<td>Sigma Aldrich</td>
			<td>GS-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Isothesia</td>
			<td>Henry Schein Animal Health</td>
			<td>29405</td>
			<td></td>
		</tr>
		<tr>
			<td>May-Grunwald Stain</td>
			<td>Sigma Aldrich</td>
			<td>MG-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell 6 well plates</td>
			<td>Falcon</td>
			<td>35 3046</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX60 Microscope</td>
			<td>Olympus</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast Plus Tissue Embedding Medium</td>
			<td>Fisher Brand</td>
			<td>23-021-400</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Conjugated IgG Armenian Hamster Isotype Control</td>
			<td>eBioscience</td>
			<td>12-4888-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered-Saline (PBS) 1x</td>
			<td>Corning cellgro</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Phycoerythrin (PE) Conjugated Anti-mouse FceRIa (clone MAR-1)</td>
			<td>eBioscience</td>
			<td>12-5898-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide Staining Solution</td>
			<td>eBioscience</td>
			<td>00-6990-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-3</td>
			<td>Peprotech</td>
			<td>213-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Safranin-o Certified</td>
			<td>Sigma Aldrich</td>
			<td>S8884</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks T25 25 cm2</td>
			<td>Beckton Dickinson</td>
			<td>353109</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks T75 75 cm2</td>
			<td>Beckton Dickinson</td>
			<td>353110</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue 1 % Aqueous</td>
			<td>LabChem-Inc</td>
			<td>LC26165-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse SCF</td>
			<td>Peprotech</td>
			<td>250-03</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing the functions of mast cells in vivo using 'mast cell knock-in' mice

乳腺细胞 （MCs） 是存在于各种组织的造血细胞，在暴露于外部环境（如皮肤、气道和胃肠道）的部位尤为丰富。以在IgE依赖过敏反应中的有害作用而闻名的MC也成为主机防御毒液和入侵细菌和寄生虫的重要参与者。MC表型和功能可能受微环境因素的影响，微环境因素可能因解剖位置和/或根据免疫反应的类型或阶段而异。因此，我们和其他人 倾向于在体内 方法而不是 体外方法， 以获得对MC函数的洞察。在这里，我们描述了小鼠骨髓衍生培养MC（BMCMCs）的生成方法，它们被收养转移到基因缺MC的小鼠，以及不同解剖部位对收养转移的MC的数量和分布的分析。这种方法，被称为"桅杆细胞敲击" 的方法，已经广泛使用在过去30年评估MC和MC衍生产品的 功能在体内。我们根据近年来开发的替代方法讨论了这种方法的优点和局限性。

图1中概述了"桅杆细胞敲击"方法，包括BMCMCs的生成、应移植到缺MC小鼠体内的细胞数量（如果根据实验设计指示，数量可以有所变化）和根据注射部位的增殖和实验之间的间隔（如果指示，此间隔也可能有所不同：例如，MC细胞质颗粒中存储的调解员的内容随着时间的增加而稳步增加。图2显示DMEM介质中含有20%WEHI-3细胞条件介质作为IL-3来源的具有代表...

Watch this Scientific Journal Video about Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice at JoVE.com

Analyzing the Functions of Mast Cells In Vivo Using &#039;Mast Cell Knock-in&#039; Mice

我们描述了体 外 衍生桅杆细胞的生成方法，它们被移植到乳腺细胞缺乏的小鼠中，以及不同解剖部位对网状桅杆细胞的表型、数量和分布的分析。此协议可用于评估桅杆细胞 在体内的功能。

使用"桅杆细胞敲击"小鼠分析体内桅杆细胞的功能

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

analyzing-functions-mast-cells-vivo-using-mast-cell-knock

Research

JoVE Journal

Immunology and Infection

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

10.1K 次观看.  Stanford University School of Medicine. 我们描述了体 外 衍生桅杆细胞的生成方法，它们被移植到乳腺细胞缺乏的小鼠中，以及不同解剖部位对网状桅杆细胞的表型、数量和分布的分析。此协议可用于评估桅杆细胞 在体内的功能。

视频: Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

科研

免疫与感染

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒：三氯生的影响不使用有机溶剂的化学效应

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

调查肥大细胞分泌颗粒;从合成到胞吐作用

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

腹膜源性肥大细胞的分离及其功能特征的 Ca2 +成像和脱粒测定

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

利用 Cas9 Ribonucleoproteins 生成人 NK 细胞的研究

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.