In vitro Explant Cultures to Interrogate Signaling Pathways that Regulate Mouse Lung Development
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Author:
Date:
2018-05-20
[Abstract] Early mouse lung development, including specification of primordia, patterning of early endoderm and determination of regional progenitor cell fates, is tightly regulated. The ability to culture explanted embryonic lung tissue provides a tractable model to study cellular interactions and paracrine factors that regulate these processes. We provide up-to-date protocols for the establishment of this culture model and its application to investigate hedgehog signaling in the developing lung.
[摘要] 早期鼠肺发育,包括原基的规范,早期内胚层的构图和区域祖细胞命运的确定,受到严格的调控。 培养移植胚胎肺组织的能力提供了一种易处理的模型来研究调节这些过程的细胞相互作用和旁分泌因子。 我们提供最新的协议,以建立这种文化模式及其应用来研究肺部发育中的刺猬信号。
【背景】小鼠肺发育起始于前肠前内胚层的内胚层憩室(E9.5),随后关闭近端气管食管中隔以形成不同的气管和食道管(Minoo和King,1994)。原始内胚层管的随后分支通过E12.5产生平面肺结构,随后正交分支产生成熟肺的三维结构特征(Metzger等人,2008)。在E12.5之前分离的肺遗传的平面结构适合于在空气液体界面进行体外培养(Carraro等人,2010; Del Moral和Warburton, 2010)。胚胎肺通过解剖使用立体显微镜在亮场照明下或通过与谱系追踪和荧光报道分子偶联时的荧光照明进行分离。在这里,我们描述了使用Shh Cre / Rosa mTmG报告小鼠,其允许Cre介导的从约E8.75起在前部前肠内胚层内的膜定位的GFP的激活(Montgomery <等人,2007;高斯等人,2009; Yao等人,2017)。因此,肺内胚层通过红色荧光由绿色荧光和周围组织显现,从而允许清晰识别和显微切割包括肺在内的发育内胚层结构,并在体外培养期间成像。
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In vitro Antigen-presentation Assay for Self- and Microbial-derived Antigens
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Author:
Date:
2017-06-05
[Abstract] Antigen presenting cells (APC) are able to process and present to T cells antigens from different origins. This mechanism is highly regulated, in particular by Patter Recognition Receptor (PRR) signals. Here, I detail a protocol designed to assess in vitro the capacity of APC to present antigens derived from bacteria, apoptotic and infected apoptotic cells.
[摘要] 抗原呈递细胞(APC)能够处理和呈递来自不同来源的T细胞抗原。这种机制是高度调节的,特别是通过Patter Recognition Receptor(PRR)信号。在这里,我详细说明了一种设计用于评估体外的APC方案,用于展示来源于细菌,凋亡和感染的凋亡细胞的抗原。
背景 T细胞淋巴细胞在其表面上表达T细胞受体(TCR),其允许识别作为与主要组织相容性复合物(MHC)分子结合的抗原加工和呈递的抗原的细胞(自身)或微生物(非自身)抗原)呈递细胞(APC)。 APC能够处理抗原并将其呈递给T细胞,并且MHC-TCR相互作用是感染和自身免疫应答期间T细胞活化的关键步骤。 &NBSP;以前的作品已经描述了基于刺激模式识别受体(PRR),例如toll样受体(TLR)(Blander和Medzhitov,2004和2006)的抗原呈递的调节机制。实际上,特异性地来自含有微生物病原体的吞噬体的TLR信号有利于在MHC-II分子内呈递非自身抗原。另一方面,凋亡细胞吞噬后产生的自身抗原由于不存在TLR刺激而导致溶酶体降解。然而,当两者都来自感染的凋亡细胞并且同时由相同的吞噬体携带时,自身和非自身抗原的分离不会发生,其由针对抗原呈递的TLR信号最佳地定制。已经使用骨髓来源的树突状细胞(BMDC)和凋亡性小鼠B细胞 - ...
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Pituitary Isograft Transplantation in Mice
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Author:
Date:
2017-06-05
[Abstract] The mouse pituitary isograft is a technique developed to administer persistent hormone stimulation, thereby increasing cellular proliferation in the mammary tissue (Christov et al., 1993). The pituitary isograft procedure was first described in ‘Induction of Mammary Cancer in Mice without the Mammary Tumor Agent by Isografts of Hypophyses’ by O. Mühlbock and L. M. Boot in 1959 (Muhlbock and Boot, 1959). Since then, the procedure has seen wide use. A pituitary gland is harvested posthumously from a donor mouse and implanted under the renal capsule of the recipient mouse through a small abdominal incision just below the last rib. Once the pituitary gland is implanted, it begins releasing hormones. These secretions increase serum levels of multiple hormones including prolactin, ...
[摘要] 小鼠垂体同种异体移植是开发用于施用持续激素刺激的技术,从而增加乳腺组织中的细胞增殖(Christov等人,1993)。垂体同种异体移植手术首先在O.Mühlbock和L.M. Boot于1959年(Muhlbock和Boot,1959)的“Inction of Mammary Cancer in Mice without the Mammary Tumor Agent by Isoplagen of Hypophyses”中被描述。此后,程序得到广泛的应用。从供体小鼠后期收获垂体腺,并通过位于最后肋骨正下方的小腹切口植入受体小鼠的肾囊下。脑垂体植入后,开始释放激素。这些分泌物增加多种激素的血清水平,包括催乳素,孕酮和17β-雌二醇(Christov等人,1993)。尽管这些激素对癌细胞增殖,生长,分化和寿命的影响没有被很好地表征,并且在一些情况下是有争议的,垂体同种移植物的净效应是根据应变特异性增加鼠乳腺组织的增殖特征(Lydon等人,1999)。 &NBSP;下面是描述如何进行垂体同种异体移植手术的协议。经过许多步骤,时间参考列在括号中。每个引用对应于该过程的嵌入式视频中的时间点。 (视频1)
背景 ...
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