Dual Fluorescence Reporter Based Analytical Flow Cytometry for miRNA Induced Regulation in Mammalian Cells
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Author:
Date:
2018-09-05
[Abstract] MicroRNA-induced gene regulation is a growing field in basic and translational research. Examining this regulation directly in cells is necessary to validate high-throughput data originated from RNA sequencing technologies. For this several studies employ luciferase-based reporters that usually measure the whole cell population, which comes with low resolution for the complexity of the miRNA-induced regulation. Here, we provide a protocol using a dual-fluorescence reporter and flow cytometry reaching single cell resolution; the protocol contains a simplified workflow that includes: vector generation, data acquisition, processing, and analysis using the R environment. Our protocol enables high-resolution measurements of miRNA induced post-transcriptional gene regulation and combined with ...
[摘要] MicroRNA诱导的基因调控是基础和转化研究中不断增长的领域。 直接在细胞中检查该调节对于验证源自RNA测序技术的高通量数据是必要的。 对于这一研究,一些研究采用基于荧光素酶的报告基因,通常测量全细胞群,其具有低分辨率的miRNA诱导调节的复杂性。 在这里,我们提供使用双荧光报告基因和流式细胞仪达到单细胞分辨率的方案; 该协议包含一个简化的工作流程,包括:使用R环境进行矢量生成,数据采集,处理和分析。 我们的协议可实现miRNA诱导的转录后基因调控的高分辨率测量,并结合系统生物学,可用于估计miRNA的熟练程度。
【背景】MicroRNAs(miRNA)是高度保守的小型非蛋白质编码RNA(21-22nt),可调节转录后基因表达并调节基因生物学过程,如发育和细胞稳态(Lagos-Quintana et al。,2001; Fabian et al。,2010; Bartel,2018),包括miRNA表达与肿瘤进展和侵袭性相关的几种病理(Lu et al。, 2005; Di Leva和Croce,2013; Krishnan et al。,2015; Bertoli et ...
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Phagocytosis Assay to Measure Uptake of Necroptotic Cancer Cells by BMDCs
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Author:
Date:
2016-11-05
[Abstract] This protocol is a flow cytometry-based method to measure the phagocytosis efficiency of necroptotic target cells by bone marrow-derived dendritic cells (BMDCs) in vitro (Aaes et al., 2016). The method is a slightly modified and updated version of the protocols used in previously published papers (Krysko et al., 2006; Brouckaert et al., 2004). In brief, the target cells are labeled with a CellTrackerTM dye before they are induced to undergo cell death. After a co-culture period of 2 h with BMDCs, the cells are immunostained with a dendritic cell marker and dead cell marker, and the phagocytic efficiency is quantified using a flow cytometer. This protocol can readily be used for target cells undergoing cell death modalities other than ...
[摘要] 该方案是基于流式细胞术的方法,以测量骨髓来源的树突细胞(BMDCs)在体外的坏死性靶细胞的吞噬效率(Aaes等人 2016)。该方法是先前发表的论文中使用的方案的稍微修改和更新的版本(Krysko等人,2006; Brouckaert等人,2004)。简言之,在细胞被诱导经历细胞死亡之前,用CellTracker TM TM染料标记靶细胞。在用BMDCs共培养2小时后,用树突细胞标记物和死细胞标记物对细胞进行免疫染色,并使用流式细胞仪定量吞噬效率。该方案可以容易地用于经历除坏死作用以外的细胞死亡模式的靶细胞。 [背景] 研究BMDCs吞噬细胞摄取的坏死细胞是检测免疫原性细胞死亡模型的初步步骤(Obeid等人,2007)。有效摄取将允许吞噬细胞将抗原交叉呈递到白细胞,从而产生针对死的靶细胞的免疫反应。在该协议中,我们使用CellTracker TM sup TM染料。这种类型的染料在某些浓度下可能是有毒的,其可以根据使用的细胞类型而变化。因此,我们建议用户首先为使用的靶细胞找到最佳的染料浓度。最佳地,CellTracker TM 染料本身不应该诱导任何细胞死亡,而是应当标记靶细胞,使得它们容易与CD11c阳性BMDC分离。
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