| A Versatile Protocol to Quantify BCR-mediated Phosphorylation in Human and Murine B Cell Subpopulations
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Author:
Date:
2021-02-05
[Abstract] Signal transduction is the process by which molecular signals are transmitted from the cell surface to its interior, resulting in functional changes inside the cell. B cell receptor (BCR) signaling is of crucial importance for B cells, as it regulates their differentiation, selection, survival, cellular activation and proliferation. Upon BCR engagement by antigen several protein kinases, lipases and linker molecules become phosphorylated. Phosphoflow cytometry (phosphoflow) is a flow cytometry-based method allowing for analysis of protein phosphorylation in single cells. Due to recent advances in methodology and antibody availability – together with the relatively easy quantification of phosphorylation – phosphoflow is increasingly and more commonly used, compared to classical ...
[摘要] [背景] 信号转导是分子信号从细胞表面传递到细胞内部的过程，导致细胞内部功能发生变化。B细胞受体（BCR）信号对于B细胞至关重要，因为它调节了它们的分化，选择，存活，细胞活化和增殖。抗原与BCR结合后，一些蛋白激酶，脂肪酶和接头分子会被磷酸化。磷酸流式细胞仪（phosphoflow）是一种基于流式细胞仪的方法，可以分析单个细胞中的蛋白质磷酸化。由于方法学和抗体的可获得性的最新进展，再加上相对容易的磷酸化定量，与传统的蛋白质印迹分析相比，越来越多地使用磷酸流。但是，建立一种适用于所有感兴趣目标的方法可能具有挑战性。在这里，我们提出了逐步的磷流方案，允许对信号分子的磷酸化状态进行评估，并进行广泛的染色，以鉴定各种人和鼠B细胞亚群，如先前在Rip等人的原始论文。，（2020 ）。在原始论文中对磷流靶标的描述旁边，我们提供了在BCR信号传导中起关键作用的其他靶标的指导。循序渐进的磷酸流操作流程是用户友好的操作，可对人和鼠B细胞亚群中各种BCR信号分子的磷酸化进行灵敏检测。
[背景] ...
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| Identification and Quantitation of Leukocyte Populations in Human Kidney Tissue by Multi-parameter Flow Cytometry
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Author:
Date:
2018-08-20
[Abstract] Inflammatory immune cells play direct pathological roles in cases of acute kidney injury (AKI) and chronic kidney disease (CKD). However, the identification and characterization of distinct populations of leukocytes in human kidney biopsies have been confounded by the limitations of immunohistochemical (IHC)-based techniques used to detect them. This methodology is not amenable to the combinations of multiple markers necessary to unequivocally define discrete immune cell populations. We have developed a multi-parameter, flow cytometric-based approach that addresses the need for panels of cell-specific markers in the identification of immune cell populations, allowing both the accurate detection and quantitation of leukocyte subpopulations from a single, clinical kidney biopsy specimen. In ...
[摘要] 炎症性免疫细胞在急性肾损伤（AKI）和慢性肾病（CKD）的病例中发挥直接的病理作用。然而，人肾活组织检查中不同白细胞群的鉴定和表征已被用于检测它们的基于免疫组织化学（IHC）的技术的局限性所混淆。该方法不适用于明确定义离散免疫细胞群所必需的多种标志物的组合。我们开发了一种多参数，基于流式细胞仪的方法，解决了在鉴定免疫细胞群体时需要细胞特异性标记组，从而允许从单个临床肾活检标本中准确检测和定量白细胞亚群。在该方法中，将新鲜的人肾组织解离成单细胞悬浮液，然后进行抗体标记和基于流式细胞术的采集和分析。这种新技术为鉴定和计数人类肾脏疾病中的免疫细胞亚群提供了重大进步，并且是补充临床肾脏活组织检查的传统组织病理学检查的有力平台。
【背景】 肾脏疾病的发病率和患病率正在上升，给全世界的医疗保健社区带来了沉重的负担，并显着影响了患者的生活质量（世界肾脏日：慢性肾病，2015年）。肾脏损害可能由多种损伤引起，包括感染，缺血，毒素，高血压，遗传和代谢紊乱（Imig和Ryan，2013）。对功能性损伤的即时反应是急性肾损伤（AKI），这是一种临床综合征，其特征在于生理上通过肾功能的快速（数小时至数天）降低和组织形态学上通过炎性免疫细胞浸润到肾小管中，组织隔室邻接小管。肾脏（Basile ...
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