| 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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| Differentiation of THP1 Cells into Macrophages for Transwell Co-culture Assay with Melanoma Cells
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Author:
Date:
2015-11-05
[Abstract] Understanding how immune cells such as macrophages interact with cancer cells is of increasing interest, as cancer treatments move towards combing both targeted- and immuno-therapies in new treatment regimes. This protocol is using THP-1 cells, a human leukemia monocytic cell line that can be differentiated into macrophages. This allows studying the effects of the macrophage secretome on cancer cells (on e.g., growth, drug response or gene expression) in co-cultures without direct cell contact interactions. This is an important aspect as it removes the presence of any phagocytic aspect to changes in the cancer cell number and behaviour. The in vitro THP-1 monocyte differentiation into polarized macrophages was used to study the effects of both M1 and M2 type populations ...
[摘要] 了解免疫细胞如巨噬细胞如何与癌细胞相互作用越来越感兴趣，因为癌症治疗趋向于在新的治疗方案中结合靶向和免疫治疗。该协议使用THP-1细胞，人类白血病单核细胞系，可以分化成巨噬细胞。这允许在没有直接细胞接触相互作用的共培养物中研究巨噬细胞分泌蛋白组织对癌细胞的作用（例如生长，药物反应或基因表达）。这是一个重要的方面，因为它消除了任何吞噬方面的癌细胞数量和行为的变化的存在。将体外THP-1单核细胞分化为极化巨噬细胞用于研究巨噬细胞的M1和M2型群体对黑素瘤细胞的作用（Smith等人，2014年， ; Tsuchiya等人，1980）。 M1型巨噬细胞通常被认为是与M2型巨噬细胞相反的肿瘤抑制，M2型巨噬细胞被认为具有组织修复和促进肿瘤生长的活性。
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| 13C Kinetic Labeling and Extraction of Metabolites from Adherent Mammalian Cells
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Author:
Date:
2015-04-20
[Abstract] Fluctuations in metabolite levels in mammalian cells are the most direct form of readout of the cellular metabolic state. The current protocol describes a method for pulse labeling and subsequent isolation of metabolites from adherent mammalian cells. The isolated metabolites can be identified and quantified by mass-spectrometry, allowing for estimation of the rates of synthesis and removal of metabolites from the system being analyzed.
[摘要] 哺乳动物细胞中代谢物水平的波动是细胞代谢状态的最直接形式的读出。 当前的协议描述了用于脉冲标记和随后从贴壁哺乳动物细胞分离代谢物的方法。 分离的代谢物可以通过质谱法鉴定和定量，允许估计代谢物从所分析的系统的合成速率和去除速率。
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