| Flow Cytometry of CD14, VDR, Cyp27 and Cyp24 and TLR4 in U937 Cells
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Author:
Date:
2020-08-05
[Abstract] Chronic Kidney Disease (CKD) patients present a micro inflammation state due to failure renal function. The calcitriol has been described as an anti-inflammatory factor that might modulates the inflammatory response in CKD patients. However, these patients have deficiency of Calcitriol due to failure renal function. But, synthesis of this vitamin has been reported in extra renal production, as in monocytes. In this context, it has been reported that the supplementation with 25 vitamin D (calcidiol or inactive form of vitamin D) induces monocytes to downregulate inflammation, due to the intracellular 1α-hidroxilase that converts calcidiol to calcitriol in these cells. Besides some reports used RT-qPCR, Western Blot or immunofluorescence techniques to investigate the expression of ...
[摘要] [摘要] 慢性肾脏病（CKD）患者由于肾功能衰竭而出现微炎症状态。骨化三醇被认为是一种抗炎因子，可能调节CKD患者的炎症反应。然而，这些患者由于肾功能衰竭而出现骨化三醇缺乏症。但是，据报道，这种维生素的合成是在肾外产生的，比如在单核细胞中。在这种情况下，有报道称，补充25维生素D（钙二醇或不活跃形式的维生素D）可诱导单核细胞下调炎症反应，这是由于细胞内的1α-羟脯氨酸酶将钙二醇转化为骨化三醇。本研究除采用RT-qPCR、Western-Blot或免疫荧光技术研究炎症和维生素D机械生物标志物在几种疾病中的表达外，本研究还应用流式细胞术技术评价25种维生素D对CD14、Toll样受体4（TLR4）、维生素D受体（VDR）的影响，单核细胞系（U937）中的1-α羟化酶（CYP27）、24-羟化酶（CYP24）。将U937培养物与健康或CKD血清孵育，用或不加25维生素D（50 ng/ml，24 h）处理，以评估CD14、TRL4、VDR、CYP27和CYP24的表达。该方案显示了研究25维生素D处理对细胞内和细胞膜生物标志物表达的影响的优势。此外，与RT-qPCR、westernblot或免疫荧光法相比，该技术不费力，但易于操作和解释。
[背景] ...
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| Imaging Cytokine Concentration Fields Using PlaneView Imaging Devices
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Author:
Date:
2018-04-05
[Abstract] We describe here a method to visualize concentration fields of cytokines around cytokine-secreting cells. The main challenge is that physiological cytokine concentrations can be very low, in the pico-molar range. Since it is currently impossible to measure such concentrations directly, we rely on cell’s response to the cytokines–the phosphorylation of a transcription factor–that can be visualized through antibody staining. Our devices aim at mimicking conditions in dense tissues, such as lymph nodes. A small number of secreting cells is deposited on a polylysine-coated glass and covered by multiple layers of cytokine-consuming. The cells are left to communicate for 1 h, after which the top layers are removed and the bottom layer of cells is antibody labeled for the response to cytokines. ...
[摘要] 我们在这里描述了一种可视化细胞因子分泌细胞周围细胞因子浓度场的方法。主要挑战是生理细胞因子浓度可能非常低，在微摩尔浓度范围内。由于目前不可能直接测量这样的浓度，我们依赖于细胞对细胞因子的反应 - 转录因子的磷酸化 - 可以通过抗体染色显现。我们的设备旨在模仿密集组织中的条件，如淋巴结。少数分泌细胞沉积在聚赖氨酸包被的玻璃上并被多层细胞因子消耗覆盖。将细胞连通1小时，之后去除顶层，并且细胞的底层被抗体标记为对细胞因子的应答。然后通过标准荧光显微镜观察细胞因子场的横截面。这篇手稿总结了我们的方法，以量化密集细胞体外细胞因子介导的细胞间通讯的程度。
【背景】哺乳动物的免疫系统已经发展到能够识别和限制潜在病原体的传播，同时使由免疫系统本身造成的附带组织损伤最小化。为了实现这一点，免疫细胞依赖细胞因子介质网络，这些细胞因子介质能够进行细胞间通讯并广播关于致病性侮辱的大小和性质的信息。大量不同细胞因子与其同源受体强烈结合，通常在纳摩尔或皮摩尔范围内具有特征性结合亲和力。通过细胞因子通讯产生免疫龛。例如，在骨髓和胸腺中，通过基质细胞分泌的白细胞介素-7（IL-7）分别支持增殖的B细胞和T细胞祖细胞的存活（Tokoyoda et al。， 2004; Alves等人，2009）。细胞因子生态位的大小控制成熟祖细胞的数量，从而保持血细胞区室平衡（Böyum，1968; ...
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| PKC-θ in vitro Kinase Activity Assay
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Author:
Date:
2016-10-20
[Abstract] Protein kinase C-θ (PKC-θ), a member of the Ca2+-independent PKC subfamily of kinases, serves as a regulator of T cell activation by mediating the T cell antigen receptor (TCR)- and coreceptor CD28-induced activation of the transcription factors NF-κB and AP-1 and, to a lesser extent, NFAT, and, subsequently, interleukin 2 (IL-2) production and T cell proliferation. In T cells, TCR and CD28 stimulation-induced activation of PKC-θ is the integrated result of diacylglycerol-mediated membrane recruitment, GLK-mediated phosphorylation at activation loop, CD28, Lck, and sumoylation-mediated central immunological synapse localization (Wang et al., 2015; Monks et al., 1997; Kong et al., 2011; Isakov and Altman, 2012; Chuang et al., 2011). ...
[摘要] Sumoylation控制许多细胞过程。蛋白激酶C-θ（PKC-θ）是激酶的Ca 2+超家族PKC亚家族的成员，通过介导T细胞抗原受体（TCR）作为T细胞活化的调节剂， - 和共受体CD28诱导的转录因子NF-κB和AP-1的活化，以及较小程度的NFAT，以及随后的白细胞介素2（IL-2）产生和T细胞增殖。我们最近证明了TCR诱导的PKC-θ的sumoylation是其在T细胞中的功能所必需的（Wang等人，2015）。在这里我们描述分析TCR诱导的过表达或内源性PKC-θ的sumoylation的方法，这是通过免疫沉淀PKC-θ进行，然后用抗SUMO1抗体进行免疫印迹。
[背景] ...
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