| A Robust Mammary Organoid System to Model Lactation and Involution-like Processes
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Author:
Date:
2021-04-20
[Abstract] The mammary gland is a highly dynamic tissue that changes throughout reproductive life, including growth during puberty and repetitive cycles of pregnancy and involution. Mammary gland tumors represent the most common cancer diagnosed in women worldwide. Studying the regulatory mechanisms of mammary gland development is essential for understanding how dysregulation can lead to breast cancer initiation and progression. Three-dimensional (3D) mammary organoids offer many exciting possibilities for the study of tissue development and breast cancer. In the present protocol derived from Sumbal et al., we describe a straightforward 3D organoid system for the study of lactation and involution ex vivo. We use primary and passaged mouse mammary organoids stimulated with fibroblast growth factor 2 ...
[摘要]
[摘要]乳腺是一种高度动态的组织，在整个生殖生活中都会发生变化，包括青春期的生长以及怀孕和进化的重复周期。乳腺肿瘤诊断代表在世界女性最常见的癌症宽。研究的监管机制乳腺的发育是至关重要的理解荷兰国际集团d如何YS调节可导致乳腺癌的发生和发展。三维（3D）乳腺组织体提供了许多令人激动的可能性的研究的组织发育和乳腺癌。在第E存在衍生自协议Sumbal等人，我们描述一个简单的3D类器官系统的研究的泌乳和复古体外。我们使用成纤维细胞生长因子2 （FGF2）和催乳素刺激的原代和传代小鼠乳腺类器官来模拟小鼠乳腺泌乳和内卷过程的三个周期。这种3D模型类器官代表一个有价值的工具来研究后期产后乳腺的发育和乳腺癌，尤其是产后-相关性乳腺癌。
图形摘要：
乳腺类器官的分离和培养程序
[背景技术]的Th e是乳腺的主要功能是提供营养吨经由牛奶产量Ò新生儿。牛逼乳腺他的发展是主要发生在出生后，由几个因素，包括激素和生长因子调控的一个高度动态的过程（Brisken和拉贾拉姆，2006;斯特恩利希特，2006年）。在青春期，激素和生长因子调节基本的胚胎导管树的导管形态发生（Brisken and ...
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| Generating Three-dimensional Human Granulomas in vitro to Study Mycobacterium tuberculosis-host Interaction
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Author:
Date:
2020-11-20
[Abstract] Granulomas are organized multicellular structures that constitute the hallmark of an infection by the human pathogen Mycobacterium tuberculosis (Mtb). A better understanding of the complex host-Mtb interactions within the granuloma’s environment may lead to new therapeutic or preventive tools to improve the control of the tuberculosis pandemic. To date, several in vitro models that are able to mimic human nascent granulomas have been reported. Here we describe a protocol in which Mtb-infected human peripheral blood mononuclear cells (PBMCs) are embedded within a collagen matrix leading to the formation of three-dimensional micro-granulomas. Subsequently, PBMCs and Mtb can be retrieved allowing multiparametric readouts from both the host and the ...
[摘要] [摘要]肉芽肿是有组织的多细胞结构，构成了人类病原体结核分枝杆菌（Mtb ）感染的标志。对肉芽肿环境中复杂的宿主-Mtb相互作用的更好理解可能会导致新的治疗或预防工具，以改善对结核病大流行的控制。迄今为止，已经报道了几种能够模仿人类新生肉芽肿的体外模型。在这里我们描述一个协议，其中Mtb 被感染的人外周血单核细胞（PBMC）嵌入胶原蛋白基质中，导致形成三维微肉芽肿。随后，可以检索PBMC和Mtb，从而从宿主和病原体中读取多参数。除了并入生理性细胞外基质外，该模型还具有重现休眠样Mtb特征的独特优势，以及在免疫调节治疗下观察到的Mtb复苏的再生，其他已发表的体外实验方法尚未见报道。肉芽肿。
关键词：分枝杆菌，浴池erculosis，肉芽肿，主机，我ñ体外模型，休眠，复苏
[背景]结核病（TB）是一种空气传播的疾病，其包括由人类病原体肺和肺外感染结核分枝杆菌（Mtb的）。结核病在2019年估计造成150万人死亡（世卫组织，2019年），仍然是世界上最致命的传染病。结核病免疫发病机制的特点是形成了组织性的，称为肉芽肿的多细胞簇（Gengenbacher和Kaufmann，2012）。这些结构主要由被感染的和未感染的巨噬细胞的核心组成，周围是淋巴细胞边缘。肉芽肿内的敌对环境推动Mtb进入可能与疾病的潜在形式相关的缓慢或非复制性休眠状态。因此，Mtb休眠导致对针对分枝杆菌复制过程中活跃的代谢途径的抗生素的耐受性增加。 ...
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| Super-resolution Imaging of the T cell Central Supramolecular Signaling Cluster Using Stimulated Emission Depletion Microscopy
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Author:
Date:
2020-11-05
[Abstract] Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center interface of T cells activated by antigen presenting cells (APC). The adaptor protein linker for activation of T cells (LAT) is a key cSMAC component. The cSMAC has widely been studied using total internal reflection fluorescence microscopy of CD4+ T cells activated by planar APC substitutes. Here we provide a protocol to image the cSMAC in its cellular context at the interface between a T cell and an APC. Super resolution stimulated emission depletion microscopy (STED) was utilized to determine the localization of LAT, that of its active, phosphorylated form and its entire pool. Agonist ...
[摘要] [摘要]超分子信号组装体因其独特的信号传导特性而受到关注。在抗原呈递细胞（APC）激活的T细胞的中心界面处形成一个微米级的信号传导组件，即中央超分子信号簇（cSMAC ）。用于激活T细胞（LAT）的衔接子蛋白接头是关键的cSMAC组件。所述CSMAC已被广泛使用的CD4全内反射荧光显微镜研究+由平面APC替代活化的T细胞。在这里，我们提供了一种协议，可以在T细胞和APC之间的接口在其细胞上下文中成像cSMAC 。超分辨率激发发射耗尽显微镜（STED）用于确定LA T的定位，其活性，磷酸化形式及其整个池的位置。在固定和抗体染色之前，将载有激动剂肽的APC与TCR转基因CD4 + T细胞孵育4.5分钟。固定的细胞对在Leica SP8 AOBS共聚焦激光扫描显微镜上使用100x 1.4 NA物镜成像。LAT聚集在多个超分子复合物中，并确定了它们的数量和大小分布。使用此协议，可以量化在T细胞和APC之间的界面在其细胞环境中的cSMAC属性。
[背景] ...
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