| Flow Cytometric Quantification of Fatty Acid Uptake by Mycobacterium tuberculosis in Macrophages
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Author:
Date:
2018-02-20
[Abstract] Mycobacterium tuberculosis (Mtb) has evolved to assimilate fatty acids from its host. However, until recently, there was no reliable way to quantify fatty acid uptake by the bacteria during host cell infection. Here we describe a new method to quantify fatty acid uptake by intracellular bacilli. We infect macrophages with Mtb constitutively expressing mCherry and then metabolically label them with Bodipy-palmitate. Following the labeling procedure, we isolate Mtb-containing phagosomes on a sucrose cushion and disrupt the phagosomes with detergent. After extensive washes, the isolated bacteria are analyzed by flow cytometry to determine the level of Bodipy-palmitate signal associated with the bacteria. Using a Mtb mutant strain defective in fatty acid uptake in liquid culture we ...
[摘要] 结核分枝杆菌（Mtb）已经发展为从其宿主吸收脂肪酸。然而，直到最近，还没有可靠的方法来量化宿主细胞感染期间细菌对脂肪酸的摄取。在这里，我们描述了一种新的方法来量化细胞内杆菌对脂肪酸的摄取。我们用Mtb组成性表达mCherry感染巨噬细胞，然后用Bodipy-palmitate代谢标记它们。标记程序后，我们在蔗糖垫上分离含有Mtb的吞噬体，并用去污剂破坏吞噬体。大量洗涤后，通过流式细胞术分析分离的细菌以确定与细菌相关的Bodipy-棕榈酸酯信号的水平。使用液体培养物中脂肪酸摄取缺陷的Mtb突变株，我们确定该突变体在巨噬细胞感染期间同化比野生型菌株少10倍的Bodipy-棕榈酸酯。脂肪酸摄取的这种定量方法可用于进一步鉴定参与细胞内Mtb和可能的其他细菌的脂质摄取的途径。
【背景】结核分枝杆菌（Mtb）同化宿主来源的脂质（脂肪酸和胆固醇）的能力使得病原体能够在其宿主内存活（Russell等人，2010; Lovewell 等人，2016）。在小鼠感染期间和在人肺组织中，通过巨噬细胞内的Mtb上调胆固醇和脂肪酸代谢相关基因来支持该想法（Schnappinger等人，2003; Rachman等人，2006; Rohde等人，2007;Fontán等人，2008; Tailleux等人，2008; Homolka et al。，2010; Rohde et ...
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| Isolation of THY1+ Undifferentiated Spermatogonia from Mouse Postnatal Testes Using Magnetic-activated Cell Sorting (MACS)
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Author:
Date:
2016-12-20
[Abstract] In mammals, homeostasis of many tissues rely on a subpopulation of cells, referred to as stem cells, to sustain an appropriate number of undifferentiated and differentiated cells. Spermatogonial stem cells (SSCs) provide the fundamental cellular source for spermatogenesis and are responsible for the lifelong maintenance of the germline pool in testes throughout the reproductive lifespan of males. To gain insight into germline stem cell biology and develop strategies for infertility treatment, several germ cell isolation methods have been reported in order to acquire good quality and quantity of undifferentiated spermatogonia. Among them, magnetic-activated cell sorting (MACS) is an efficient cell isolation method that requires less time and less initial cell numbers to obtain an enriched ...
[摘要] 在哺乳动物中，许多组织的体内平衡依赖于称为干细胞的细胞亚群，以维持适量的未分化细胞和分化细胞。精原细胞干细胞（SSC）为精子发生提供了基本的细胞来源，并且负责终生维持雄性生殖期间睾丸中的种系池。为了获得对种系干细胞生物学的了解并制定不孕症治疗策略，已经报道了几种生殖细胞分离方法，以获得良好质量和数量的未分化精原细胞。其中，磁激活细胞分选（MACS）是一种有效的细胞分离方法，其需要较少的时间和较少的初始细胞数以使用抗原 - 抗体反应来获得富集的细胞群体。胸腺细胞抗原1（THY1，CD90.2）被认为是小鼠新生儿和成年睾丸中未分化精原细胞的表面标志物。在这里，我们描述了一个用于分离来自小鼠睾丸的一周龄THY1 + 细胞和四周龄THY1 + 细胞的方案。分离过程包括三个步骤：睾丸收集和单细胞悬浮，使用生物素缀合的抗THY1抗体的细胞标记和磁性细胞分离。注意，这种隔离方案应在五小时内完成，以最大限度地提高活细胞的质量和数量。
背景 在几个成人组织中观察到活性和静止干细胞的共存。静止，自我更新和分化之间的充分平衡对于维持适当数量的未分化干细胞是必要的，并且避免过早的干细胞耗尽许多组织的体内平衡（Tseng等人，2015; Wabik和Jones，2015; ...
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