| A Modified Semisolid Clonal Culture for Identification of B-1 and B-2 Progenitor Colony Forming Ability of Mouse Embryonic Hemogenic Endothelial Cells
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Author:
Date:
2020-05-05
[Abstract] The search for the origin of the first hematopoietic stem cells (HSCs) in the mouse embryo has been a hot topic in the field of developmental hematopoiesis. Detecting lymphoid potential is one of the supportive evidence to show the definitive hematopoietic activity of HSCs. However, the first B-lymphoid potential in the mouse embryos are reported to be biased to innate-like B-1 cell lineage that can develop from hemogenic endothelial cells (HECs) independently of HSCs. On the other hand, conventional adaptive immune B cells (B-2) cells are considered to be exclusively derived from HSCs. Therefore, segregating B-1 and B-2 progenitor potential is important to understand the developmental process of HSCs that are also produced from HECs through intermediate precursors referred to as ...
[摘要] [摘要 ] 在搜索起源的第一造血干细胞（HSCs）在小鼠胚胎一直是一个热门话题在该领域发展造血功能。检测淋巴潜力是一个支持性证据显示权威造血活动的造血干细胞然而，据报道，小鼠胚胎中的第一个B淋巴样电位偏向于先天性B-1细胞谱系，该谱系可以从造血内皮细胞（HEC）脱离HSC发育而来。 B细胞（B-2）细胞被认为仅来自HSC。因此，分离B-1和B-2祖细胞的潜力对于理解也由HEC通过中间前体（也称为HEC）产生的HSC的发育过程非常重要。 HECs和pre-HSCs均显示pre-HSCs显示内皮表面表型并需要基质支持以检测其造血活性。利用基质细胞培养后再进行改良的半固体克隆培养的方法使我们能够检测B-1的菌落形成单位数量/ B-2祖细胞最初源自HEC / HSC之前的细胞，将反映B-1偏倚或多谱系繁殖的HSC的潜力。
[背景 ] 半固体克隆培养（甲基纤维素集落形成测定法）是检测造血祖细胞数量的传统方法。一个集落被认为是来自单个祖细胞（克隆来源），添加的细胞因子在细胞的形成中起着重要作用。产量菌落，例如Epo增强了红细胞的菌落形成单位（CFU-E）或红细胞的爆发形成单位（BFU-E），而G-CSF / GM-CSF将增强CFU-G（粒细胞），M ...
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| Long-term in vitro Culture of Cryptosporidium parvum
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Author:
Date:
2018-08-05
[Abstract] Continuous in vitro growth of Cryptosporidium parvum has proved difficult and conventional in vitro culture techniques result in short-term (2-5 days) growth of the parasite resulting in thin-walled oocysts that fail to propagate using in vitro cultures, and do not produce an active infection using immunosuppressed or immunodeficient mouse models (Arrowood, 2002). Here we describe the use of hollow fiber bioreactors (HFB) that simulate in vivo conditions by providing oxygen and nutrients to host intestinal cells from the basal surface and permit the establishment of a low redox, high nutrient environment on the apical surface. When inoculated with 105 C. parvum (Iowa isolate) oocysts the bioreactor produced 108 ...
[摘要] Cryptosporidium parvum 的连续体外生长已证明是困难的，并且常规体外培养技术导致短期（2-5天）生长寄生虫导致薄壁卵囊不能使用体外培养物繁殖，并且不使用免疫抑制或免疫缺陷小鼠模型产生活跃感染（Arrowood，2002）。在这里，我们描述了中空纤维生物反应器（HFB）的使用，通过提供氧气和营养物质从基础表面宿主肠细胞模拟体内条件，并允许建立低氧化还原，高营养环境顶面。当接种10 5 C时。 parvum （爱荷华州分离物）卵囊生物反应器在14天后每ml产生10个 8 卵囊（20ml额外毛细血管体积），并保持2年以上。使用TCR-α免疫缺陷小鼠模型的体内感染性研究显示，在6,12和18个月时从生物反应器产生的卵囊与用于启动培养的亲本Iowa分离物无法区分。 HFB产生的卵囊具有与亲本爱荷华分离物类似的百分比分析。
【背景】 Cryptosporidium parvum 是人和其他哺乳动物肠道的细胞内专性寄生虫，导致急性腹泻。该疾病在免疫功能正常的个体中是自限性的，然而，在免疫功能低下的成人和幼儿中，该疾病可能危及生命（Kotloff，2017）。它是经济资源低的国家中三种被诊断出的儿童肠道疾病之一（Kotloff et al。，2013; Sow et ...
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| 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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