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，甚至可以抑制某些免疫反应29，34，40。
在我们的协议描述中，我们决定专注于骨髓衍生培养的MC（BMCMCs）的生成和移植，因为大量的这些细胞可以在体外产生来自野生类型或突变小鼠的骨髓。然而，MC也可以直接从胚胎干细胞（胚胎干细胞衍生的培养MC[ESCMCs]）41培养，当基因兼容时，这些细胞也可以用于移植到缺乏MC的小鼠。这种替代方法对于研究一种蛋白质的作用特别有趣，这种蛋白质的缺乏会导致小鼠的胚胎杀伤力，因此不能产生这种蛋白质的BMCMC。在将这些细胞移植到缺乏MC的老鼠31，33之前，BMCMC和ESCMC也可以在体外用扁豆病毒编码感兴趣的基因或shRNA来沉默感兴趣的基因。
我们通常使用 20% 的 WEHI-3 条件介质作为 BMCMC 文化的 IL-3 来源。然而，重组IL-3（10 ng/ml，如步骤1.2.1所述）也可以使用，在培养介质中加入重组干细胞因子（SCF）可以大大增加BMCMC产生的42，43个。根据研究，除了IL-3外，还使用了10至100 ng/ml的重组SCF来产生BMCMC36，44，45。人们应该记住，来自不同供应商的商用穆林重组SCF制剂在影响BMCMC发展方面可能有不同的效力。同样重要的是要认识到，用于生成BMCMC的方法细节（例如是否将重组SCF添加到IL-3含介质、培养期的持续时间 等）可能会影响此类细胞的表型和功能。例如，据报道，长期接触SCF的BMCMC增加了组胺和某些蛋白酶44，46的水平，但在 体外45显示FcεRI介导的脱粒和细胞因子生产明显减弱。最后，除了IL-3，WEHI-3条件介质包含许多可能影响MC功能的生物活性分子。因此，通过WEHI-3条件介质获得的BMCMC可能与通过重组IL-3（或重组IL-3加SCF）获得的BMCMC有所不同。关于不同类型培养介质中产生的BMCMC表型中是否存在这种差异，在细胞在 体内进入不同的解剖部位后，是否保留下来的研究很少，而且这种类型的其他研究可能也令人感兴趣。但是，无论选择的文化条件如何，应使用相同的培养媒介配方生成所有 BMCMC，用于在实验中将结果汇集起来进行分析。此外，MC应在它们进入缺乏MC的老鼠之前至少培养4至6周，以达到95-98%的纯度（图2）。这是为了减少在"BMCMC种群"中，除致力于乳腺细胞谱系的造血细胞（在将骨髓细胞置于 体外后，这些细胞在培养物中处于早期间隔）之外，还可能导致在"乳腺细胞敲击"小鼠中出现供体衍生细胞的可能性。
我们在这里提出了一个详细的协议，用于将 MC 缺陷小鼠与野生类型或突变的 BMCMCs 在内部 （即 p.）， 静脉注射 （即） 或内皮纳 （即） 在耳针， 因为这些注射途径已被许多调查人员使用。然而，BMCMCs也成功地植根于后皮47，在脚垫48，关节内49 或颅内50，51。要移植的BMCMC数量，以及移植和实验之间的间隔，可能因注射路线和目标器官的移植而异（图1）。在开始实验之前，在将BMCMC灌入后，尊重这种间隔是非常重要的，以便让BMCMC（尚未完全成熟的MC） 在体内变得更加成熟。由于储存在MC细胞质颗粒中的调解员在细胞寿命38，52的过程中可以继续增加，因此对于某些实验，人们不妨增加MC增生和启动实验之间的间隔，以评估MC功能。
根据注射路线和/或注射的BMCMCs的数量，收养转移的MC的数量和/或解剖分布可能不同于野生类型小鼠12，23，53，54的相应原生MC种群的数量和/或解剖分布。MC缺乏的老鼠与野生型小鼠的原生MC种群，在腹腔和间膜中，以及在真皮中，分别在MC转移12，23周后评估4至8周时，可以拥有与原生MC种群大致相同的数量和分布。BMCMC 的静脉输液不会导致大多数组织中的正常 MC 编号和/或分布。例如，在这种i.v.-engraft的"桅杆细胞敲击"小鼠的皮肤中，没有或很少发现MC。在将BMCMC注射到缺 MC 小鼠的 4-28 周后，气管中的 MC 数量大大低于相应野生类型的小鼠。相比之下，肺外围的MC数量通常大于相应的野生型小鼠12，23，53，55。I.v. BMCMCs的转移也导致脾脏中的MC水平很高，而很少本地MC通常发现在这个器官在野生类型的小鼠23，56。重要的是，先前的报告表明，即将BMCMC注射到缺乏MC的小鼠体内，不会导致MC人群在特定的解剖部位，如脊髓、淋巴结或心脏54，57。一些团体还指出，这种（即包扎）不会导致肠道粘性MC（MMC）人口23，58-60的侵占。在解释使用MC"桅杆细胞敲击"模型9获得的数据时，必须考虑到MC数字和/或采用转移的MC与本地MC的分布差异。
存在几种缺 MC 的鼠标菌株，选择在特定项目中使用哪一个菌株非常重要。c-套件突变 MC 缺陷小鼠，如套件W/W-v或套件W-sh/W-sh小鼠，传统上被许多研究者使用。然而，这些小鼠除了严重缺氧（表1）外，还患有许多与c-套件相关的表型异常。近年来，据报道，几株具有c-套件独立构成MC缺乏症的小鼠已报告24-26。其中一些小鼠在MC缺乏症（表1）旁边也表现出其他表型异常，而且由于这些新描述的菌株的表型仍在调查中，也可能发现其他异常。所有这些小鼠和一些额外的新型MC缺乏小鼠最近已详细审查9，10，13。
鉴于目前可用的每个 MC 缺陷菌株的局限性，我们建议尝试使用多个 MC 缺陷9模型来测试有关 MC 功能的假设。在我们的实验室中，我们通常在 套件W-sh/W-sh 和 Cpa3-Cre 中进行试点实验:麦克尔-1fl/fl 小鼠。如果我们在这两种缺 MC 小鼠中都获得一致的结果，那么我们就会进行移植实验，以确定 MC 的作用并评估某些 MC 衍生产品的潜在作用。最后，应该指出的是，一些菌株允许可诱导耗尽的MC或Cre重组介导删除"氟化物"基因在MC最近也被描述为25，49，61。这些菌株已在其他地方进行了详细的审查9，10，13， 并代表有希望的替代-或补充-方法来研究MC功能 在体内。

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">
	<tbody>
		<tr>
			<td>1% Antibiotic-Antimycotic Solution</td>
			<td>Corning cellgro</td>
			<td>30-004-Cl</td>
			<td></td>
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			<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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			<td>Alcian Blue 8GX</td>
			<td>Rowley Biochemical Danver</td>
			<td>33864-99-2</td>
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			<td>Allegra 6R Centrifuge</td>
			<td>Beckman</td>
			<td></td>
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			<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>
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			<td>BD 1 ml TB Syringe</td>
			<td>BD Syringe</td>
			<td>309659</td>
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			<td>BD 22G x1 (0.7 mm x 25 mm) Needles</td>
			<td>BD Precision Glide Needle</td>
			<td>205155</td>
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			<td>BD 25G 5/8 Needles</td>
			<td>BD Syringe</td>
			<td>305122</td>
			<td></td>
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			<td>BD 30G x1/2 Needles</td>
			<td>BD Precision Glide</td>
			<td>305106</td>
			<td></td>
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			<td>Blue MAX Jr, 15 ml Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
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		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C298-500</td>
			<td></td>
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		<tr>
			<td>Cytoseal 60 Mounting Medium</td>
			<td>Richard-Allan Scientific</td>
			<td>8310-4</td>
			<td></td>
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		<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>
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		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>F79-500</td>
			<td></td>
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		<tr>
			<td>Giemsa Stain Modified</td>
			<td>Sigma Aldrich</td>
			<td>GS-1L</td>
			<td></td>
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		<tr>
			<td>Isothesia</td>
			<td>Henry Schein Animal Health</td>
			<td>29405</td>
			<td></td>
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		<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>
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		<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>
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			<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来源的具有代表性的流动细胞学分析和BMCMC1、15和45天培养后的甲状腺素蓝色染色。请注意，培养45天的BMCMC是95%纯净的，含有大量的细胞质颗粒，可用于移植实验，而培养15天的细胞不适合移植。图3显示具有代表性的成功移植后4周的耳针（图3A），在间皮窗（图3B）和腹腔（图3C）6周后i.p.镶缩，并在肺（图3D）12周后，即怀孕。
<img alt="Figure 1" src="/files/ftp_upload/52753/52753fig1.jpg"> 图1:用于分析体内MC功能的"桅杆细胞敲击"鼠标模型。野生类型或突变骨髓衍生培养的MC（BMCMCs）是由培养骨髓细胞在20%WEHI-3细胞条件介质中至少4-6周作为IL-3的来源（或在含有10 ng/ml重组小鼠IL-3的介质中产生）。然后，这些BMCMC可以并入MC缺陷小鼠体内，以制造所谓的"桅杆细胞敲击"小鼠。BMCMC可以通过不同的途径（静脉注射[即]，内皮[即]或皮内[i.d.]）注射，用于局部（即，即）或系统性（即）对各种MC人群的重组。然后，通过比较野生小鼠、MC缺陷小鼠和"桅杆细胞敲击"小鼠的反应，可以分析各种生物反应中的MC功能。可以通过比较与野生型BMCMC或BMCMCs相植的"桅杆细胞敲击"小鼠的反应来分析特定MC产品的贡献，这些小鼠缺乏或表达基因改变的这种产品形式。（这是图1的修改版本，来自参考12）。
<img alt="Figure 2" src="/files/ftp_upload/52753/52753fig2.jpg"> 图2:通过流动细胞学和显微镜评价BMCMC制剂的纯度。 （A） 代表流动细胞学分析1天（左面板）、15天（中间面板）和45天（右面板）旧BMCMC表面的FcεRIα和KIT（CD117）表达。数字表示在蓝色正方形中封闭的FcεRIα+KIT+ BMCMC 的百分比。（B） BMCMC 代表图片染有甲苯甲酸蓝色，下面板是黑色虚线定义的上面板区域的放大倍率。酒吧=10微米。
<img alt="Figure 3" src="/files/ftp_upload/52753/52753fig3.jpg"> 图3:评估各种解剖部位的MC移植效率。（A） 代表图片的4μm耳针部分沾染了甲苯丙酸蓝色。（B） 代表照片的美森特式窗户沾染了萨夫拉宁/阿西安蓝（"Csaba"污渍）。（C） 存在腹膜熔岩液中细胞的代表图片，并沾染了梅-格伦瓦尔德·吉姆萨（MGG）。（D） 4μm 肺部沾染为血脂蓝的代表图片。条形 = 50μm （A、B和D）或 20μm（C）。图片来自 C57BL/6 野生型小鼠（上面板）、MC 缺陷套件W-sh/W-sh小鼠（中面板）和 MC 缺陷小鼠，它们与野生类型 BMCMC:BMCMCs套件W-sh/W-sh（下面板）。用箭头表示MC。
<table border="1" cellpadding="0" cellspacing="0"><tbody>
<tr>
<td>小 鼠</td>
			<td>可用背景</td>
			<td>
MC 编号 （稳定状态）</td>
			<td>
其他表型 （在9，10，13中回顾）</td>
			<td>报告与BMCC的侵联</td>
		</tr>
<tr>
<td>套件W/W-v</td>
			<td>
（WB/Re x C57BL/6）F1 （杰克逊实验室– WBB6F1/J-基特W/基特沃-v/J）</td>
			<td>缺乏结缔组织和粘性MC</td>
			<td>贫血、低基酚和中性粒细胞数量减少、黑色素细胞和卡哈尔、无菌等间质细胞的缺陷。</td>
			<td>i.v.35，37;i.p.31，62;即28，29;i.c50，51;即49:fp.i.63
</td>
		</tr>
<tr>
<td rowspan="3">套件W-嘘/W-什</td>
			<td>
C57BL/6 （杰克逊实验室– B6.在杰克逊实验室进行的单核苷酸多态性分析表明，这些小鼠只有+87%的C57BL/6遗传背景。</td>
			<td rowspan="3">缺乏结缔组织和粘性MC</td>
			<td rowspan="3">巴索菲尔和中性粒细胞数量适度增加，骨髓衍生抑制细胞数量增加64个，黑色素细胞和Cajal间质细胞缺乏</td>
			<td rowspan="3">i.v.23，34-36;i.p.31，62;即28，29;即49
</td>
		</tr>
<tr>
<td>
C57BL/6J62 （杰克逊实验室– B6.Cg-KitWsh/H尼尔贾布斯姆格利杰）这些小鼠在C57BL/6J背景上已经回过11次>。</td>
		</tr>
<tr>
<td>
巴尔布/c65 （C.B6-套件W-什）</td>
		</tr>
<tr>
<td>
麦克普特5-克雷; R-DTA
</td>
			<td>
C57BL/625 （Tg（Cma1-cre）阿罗尔:B6.129P2-格特（罗莎）26索特姆1（DTA）Lky/J）</td>
			<td>腹内科显著减少 （98%）和皮肤 （89-96.5%）Mcs， 粘性 Mcs 不太可能耗尽</td>
			<td>粘性MC的可能存在;记者小鼠透露， 在 ~30% 脾脏 Nk 细胞66中， Cre 介因删除了 "浮出" 的 Yfp 转基因
</td>
			<td>没有</td>
		</tr>
<tr>
<td rowspan="2">Cpa3克雷/+</td>
			<td>
C57BL/626 （B6.129P2-Cpa3tm3（英亩）小时）</td>
			<td rowspan="2">缺乏结缔组织和粘性MC</td>
			<td rowspan="2">
Cpa3 表示在其他细胞类型:减少嗜血者数量</td>
			<td rowspan="2">i.v.26
</td>
		</tr>
<tr>
<td>
巴尔布/c26 （B6.129P2/奥拉赫德.BALB/c-Cpa3tm3（英亩）小时）</td>
		</tr>
<tr>
<td>
Cpa3-克雷; 麦克尔-1 fl/fl
</td>
			<td>
C57BL/624 （Tg（帕3-克雷）3Gli:B6:129-麦克尔1tm3sjkJ）</td>
			<td>缺乏结缔组织和粘性MC</td>
			<td>
Cpa3 表示在其他细胞类型:脾脏中性粒细胞增加和轻度贫血; 减少嗜血者数量</td>
			<td>i.d.24: 即49 i.v. （我们未发布的数据）</td>
		</tr>
</tbody></table>表1:具有构成MC缺陷的老鼠菌株。有几种小鼠具有 c- 套件依赖或 c- 套件- 独立构成 MC 缺陷。原则上，所有这些菌株都可用于生成"桅杆细胞敲击"小鼠（不过，据我们所知，Mcpt5-Cre尚未报告:R-DTA鼠标）。然而，这些菌株中的每一个都有其他表型异常和局限性，在解释这些小鼠获得的结果时，应牢记这些异常和局限性。在参考文献中可以找到一些成功的 MC 灌接示例。（这是参考9表1的修改和更新版本 ）。

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 'Mast Cell Knock-in' 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 ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

10.2K 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, ...

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 ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

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.

使用"桅杆细胞敲击"小鼠分析体内桅杆细胞的功能

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利用 Cas9 Ribonucleoproteins 生成人 NK 细胞的研究

葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗

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