| Isolation and High Throughput Flow Cytometric Apoptosis Assay of Human Neutrophils to Enable Compound Library Screening
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Author:
Date:
2020-06-05
[Abstract] The study of human neutrophils in vitro is challenging due to their short half-life and propensity for activation. However, with careful handling and manipulation in the laboratory, they can be a powerful tool to investigate immune responses in health and disease. Here we describe a method for the isolation of human neutrophils from peripheral blood samples, followed by a high-throughput screen to assess the efficacy of a library of compounds in inducing neutrophil apoptosis, which may have therapeutic potential in neutrophil-driven diseases. This protocol is based on previously-published neutrophil isolation methods utilizing Dextran sedimentation of red blood cells followed by the separation of granulocytes with plasma/Percoll discontinuous gradient centrifugation. Yields of ~1 ...
[摘要] [摘要] 人类嗜中性粒细胞的研究 由于其半衰期短且易于活化，因此体外具有挑战性，然而，通过在实验室中的仔细处理和操纵，它们可以成为研究健康和疾病中免疫反应的有力工具。在此，我们介绍了一种分离方法外周血样本中的人类嗜中性粒细胞，然后进行高通量筛选，以评估化合物库诱导嗜中性粒细胞凋亡的功效，该化合物在嗜中性粒细胞驱动的疾病中可能具有治疗潜力。此规程基于先前发表的嗜中性粒细胞分离方法利用红细胞的葡聚糖沉降，接着用等离子体/粒细胞的分离的Percoll 〜1×10个的不连续梯度centrifugation.Yields 6 每嗜中性粒细胞毫升的血液，和>的纯度95个％嗜中性粒细胞是典型的。嗜中性粒细胞与经处理的激酶抑制剂文库，然后通过流式细胞仪评估中性粒细胞凋亡的速率。低通量用于高通量筛选人类原代免疫细胞，以鉴定具有修饰嗜中性粒细胞功能的化合物，并可根据需要进行修饰以评估其他表型。
[背景] 中性粒细胞是重要的先天免疫细胞与键的角色防御抵抗infection.They是短暂的细胞中，在感染和inflammat部位延长6-8小时循环典型的半衰期离子（萨默斯等人。 。，2010 ; Hidalgo。Et Al ...
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| 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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