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Author:
Date:
2020-12-05
[Abstract] Natural killer (NK) cells are innate immune cells, characterized by their cytotoxic capacity, and chemokine and cytokine secretion upon activation. Human NK cells are identified by CD56 expression. Circulating NK cells can be further subdivided into the CD56bright (~10%) and CD56dim NK cell subsets (~90%). NK cell-like cells can also be derived from human induced pluripotent stem cells (iPSC). To study the chemokine and cytokine secretion profile of the distinct heterogenous NK cell subsets, intracellular flow cytometry staining can be performed. However, this assay is challenging when the starting material is limited. Alternatively, NK cell subsets can be enriched, sorted, stimulated, and functionally profiled by measuring secreted effector molecules in the supernatant by Luminex. Here, ...
[摘要] [摘要]天然杀伤（NK）细胞是先天性免疫细胞，其特征在于其细胞毒性能力以及活化后的趋化因子和细胞因子分泌。人NK细胞通过CD56表达鉴定。循环的NK细胞可进一步细分为CD56亮（约10％）和CD56暗NK细胞亚群（约90％）。NK细胞样细胞也可以源自人诱导的多能干细胞（iPSC）。为了研究不同的异源NK细胞亚群的趋化因子和细胞因子分泌概况，可以进行细胞内流式细胞仪染色。然而，当起始原料有限时，该测定法具有挑战性。或者，可以通过Luminex测量上清液中分泌的效应子分子来富集，分选，刺激和功能性分析NK细胞亚群。在这里，我们提供了一种快速直接的方案，用于分离和刺激原代NK细胞或iPSC衍生的NK细胞样细胞，并随后检测分泌的细胞因子和趋化因子，这也适用于少量细胞。
[背景]自然杀伤（NK）细胞是先天免疫系统的一部分，提供第一线防御病毒感染和畸形。在人血中，可以基于CD56和CD16表达鉴定出两个不同的NK细胞群体：CD56明亮的CD16 +/-和CD56暗的CD16 + ...
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Author:
Date:
2014-05-20
[Abstract] As for all positive strand RNA viruses, hepatitis C virus (HCV) RNA replication is tightly associated with rearranged host cell membranes, termed viral replication factories. However, up to now little is known about both viral and cellular constituents of viral replication factories. Here, we describe a protocol to specifically isolate HCV-remodeled host cell membranes and endoplasmic reticulum (ER) membranes of naïve cells, by using a functional NS4B HA-tagged subgenomic replicon and a C-terminally HA-tagged calnexin-overexpressing cell line, respectively. Post-nuclear whole cell membrane fractions are first enriched by density gradient centrifugation, followed by HA-specific affinity tag purification. Upon elution under native conditions, purified samples can be subject to a variety of ...
[摘要] 至于所有正链RNA病毒，丙型肝炎病毒（HCV）RNA复制与称为病毒复制工厂的重排宿主细胞膜紧密相关。 然而，到目前为止对病毒复制工厂的病毒和细胞成分了解甚少。 在这里，我们描述一个协议，通过使用功能NS4B HA标记亚基因组复制子和C末端HA标记calnexin过表达细胞系，特异性隔离HCV重塑的宿主细胞膜和内质网（ER）膜的幼稚细胞， 分别。 首先通过密度梯度离心富集核后全细胞膜级分，随后通过HA特异性亲和标签纯化。 在天然条件下洗脱时，纯化的样品可进行多种生物化学和功能测定。
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