| Improved Macrophage Isolation from Mouse Skeletal Muscle
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Author:
Date:
2021-04-20
[Abstract] Macrophages are a heterogeneous class of innate immune cells that offer a primary line of defense to the body by phagocytizing pathogens, digesting them, and presenting the antigens to T and B cells to initiate adaptive immunity. Through specialized pro-inflammatory or anti-inflammatory activities, macrophages also directly contribute to the clearance of infections and the repair of tissue injury. Macrophages are distributed throughout the body and largely carry out tissue-specific functions. In skeletal muscle, macrophages regulate tissue repair and regeneration; however, the characteristics of these macrophages are not yet fully understood, and their involvement in skeletal muscle aging remains to be elucidated. To investigate these functions, it is critical to be able to efficiently ...
[摘要] [摘要]巨噬细胞是异质类,提供防御的在主体上的主线路先天免疫细胞的通过phagocyt定义的病原体,消化荷兰国际集团它们,本荷兰国际集团的抗原的T和B细胞以引发适应性免疫。通过专门的促炎或抗炎活动,巨噬细胞也直接有助于清除感染和修复组织损伤。巨噬细胞分布在全身,并主要执行组织特定功能。在骨骼肌中,巨噬细胞调节组织的修复和再生。H 然而,这些巨噬细胞的特征还没有被完全理解,它们在骨骼肌衰老中的作用还有待阐明。为了研究这些功能,至关重要的是能够有效地从骨骼肌中分离出足够的纯度和产量的巨噬细胞用于各种下游分析。在这里,我们详细描述了一种从小鼠中分离骨骼肌巨噬细胞的优化方法。这种方法已允许的isolat的离子的高纯度CD45 + / CD11b的+从年轻和年老小鼠,其可以进一步用于流巨噬细胞cytometr ÿ分析,荧光激活细胞分选(FACS),和单细胞RNA测序。
[背景] Metchnikoff及其同事在一个多世纪以前发现巨噬细胞为“专业”吞噬细胞(Underhill等,2016)。后来的研究表明,巨噬细胞构成一类异质的细胞,在整个人体的组织中发挥着不同的功能(Wynn等人,2013)。巨噬细胞可分为两种主要类型:组织驻留巨噬细胞和非组织驻留巨噬细胞(Ginhoux和Guilliams ...
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| Dissecting the Rat Mammary Gland: Isolation, Characterization, and Culture of Purified Mammary Epithelial Cells and Fibroblasts
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Author:
Date:
2020-11-20
[Abstract] With the advent of CRISPR-Cas and the ability to easily modify the genome of diverse organisms, rat models are being increasingly developed to interrogate the genetic events underlying mammary development and tumorigenesis. Protocols for the isolation and characterization of mammary epithelial cell subpopulations have been thoroughly developed for mouse and human tissues, yet there is an increasing need for rat-specific protocols. To date, there are no standard protocols for isolating rat mammary epithelial subpopulations. Analyzing changes in the rat mammary hierarchy will help us elucidate the molecular events in breast cancer, the cells of origin for breast cancer subtypes, and the impact of the tumor microenvironment. Here we describe several methods developed for 1) rat mammary ...
[摘要] [摘要]随着CRISPR-Cas的出现以及能够轻松修饰各种生物的基因组的能力,越来越多地开发大鼠模型来询问乳腺发育和肿瘤发生的遗传事件。已经为小鼠和人类组织彻底开发了用于分离和表征乳腺上皮细胞亚群的方案,但是对大鼠特异性方案的需求却在不断增长。迄今为止,还没有用于分离大鼠乳腺上皮亚群的标准方案。分析大鼠乳腺层次的变化将有助于我们阐明乳腺癌中的分子事件,乳腺癌亚型的起源细胞以及肿瘤微环境的影响。在这里,我们描述为1)大鼠乳腺上皮细胞分离开发的几种方法;2)大鼠乳腺成纤维细胞分离;3)培养大鼠乳腺上皮细胞;通过4)流式细胞仪分析和鉴定大鼠乳腺细胞;5)免疫荧光。源自该协议的细胞可用于多种目的,包括RNAseq ,药物研究,功能测定,基因/蛋白质表达分析和图像分析。
[背景]大多数与乳腺有关的研究都是在小鼠模型和人体样品中进行的。然而,由于其具有类似于人的药代动力学特征和乳腺发育,该疾病的大鼠模型正变得越来越流行(Russo等人,1990;Jiunn等人,2008; Smalley等人,2016)。像人类腺癌一样,大鼠乳腺癌也经历组织学发展阶段(Russo等,1990; Singh等,2000),并且是卵巢激素依赖性的(Thompson等,1998; ...
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| Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors
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Author:
Date:
2020-05-20
[Abstract] Ectopic expression of transcription factor combinations has been recently demonstrated to reprogram differentiated somatic cells towards the dendritic cell (DC) lineage without reversion to a multipotent state. DCs have the ability to induce potent and long-lasting adaptive immune responses. In particular, conventional type 1 DCs (cDC1s) excel on antigen cross-presentation, a critical step for inducing CD8+ T cell cytotoxic responses. The rarity of naturally occurring cDC1s and lack of in vitro methodologies for the generation of pure cDC1 populations strongly hinders the study of cDC1 lineage specification and function. Here, we describe a protocol for the generation of induced DCs (iDCs) by lentiviral-mediated expression of the transcription factors PU.1, IRF8 and ...
[摘要] [摘要] 转录因子组合的异位表达最近被证明可以将分化的体细胞重编程为树突状细胞(DC)谱系,而不会回复到多能状态。DC具有诱导有效和持久的适应性免疫应答的能力。在特定的常规类型1的DC(cDC1s)练成抗原交叉呈递,用于诱导CD8的关键步骤+ T细胞的细胞毒性应答。天然存在的cDC1的稀有性和缺乏用于生成纯cDC1群体的体外方法论,严重阻碍了对cDC1谱系规格和功能的研究。在这里,我们描述了用于生成感应DC(iDC)的协议 慢病毒介导的转录因子PU.1,IRF8和BATF3在小鼠胚胎成纤维细胞中的表达。iDC 在9天内获得DC形态,cDC1表型和转录特征。使用此协议生成的iDC 具有对炎症刺激,吞噬死细胞,将抗原加工并交叉呈递给CD8 + T细胞的功能。DC重新编程提供了一个简单易处理的系统,可以生成大量的cDC1类细胞用于高内涵筛选,从而开辟了新途径,可以更好地了解cDC1的规格和功能。将来,在成纤维细胞中忠实诱导cDC1命运可能会导致产生患者特定的疫苗接种DC。
[背景技术树突状细胞(DC)是专业的抗原呈递细胞,专门用于识别,加工和呈递T细胞抗原,在诱导适应性免疫应答和免疫记忆中起关键作用(Me rad 等,2013)。DC可以分为4个主要子集:浆细胞样DC(pDC ),大量1型干扰素的产生者,循环单核细胞衍生的单核细胞衍生DC 和常规DC(cDC ...
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