使用基于图像的流式细胞术与基于荧光原位杂交的FlowRNA分析方法，同时检测脂多糖刺激后单核细胞内的肿瘤坏死因子-α（TNF-α）蛋白和信使核糖核酸（mRNA）。

Using Image-Based Flow Cytometry with a FISH-Based FlowRNA Assay to Simultaneously Detect Intracellular TNF-α Protein and mRNA in Monocytes Following LPS Stimulation.

作者信息

Venable Adam S, Henning Andrea L, Prado Eric A, McFarlin Brian K

机构信息

Applied Physiology Laboratory, University of North Texas, 1155 Union Circle, Denton, TX, USA.

Department of Biological Sciences, University of North Texas, Denton, TX, USA.

出版信息

Methods Mol Biol. 2016;1389:139-51. doi: 10.1007/978-1-4939-3302-0_9.

DOI:10.1007/978-1-4939-3302-0_9

PMID:27460242

Abstract

Existing methods of assessing monocyte inflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) response to in vitro lipopolysaccharide (LPS) stimulation lack the ability to simultaneously detect intracellular mRNA and protein. This procedure takes advantage of new methodologies and instrumentation to simultaneously measure intracellular TNF-α mRNA and protein in CD14(+) monocytes after 1, 3, and 6 h of LPS stimulation. By assessing multiple timepoints, we are able to discern how LPS stimulation affects the temporal relationship between TNF-α mRNA and protein. By using image-based flow cytometry it is possible to co-localize mRNA and protein signals to identify the length of incubation that is needed to initiate protein translation.

摘要

现有的评估单核细胞炎性细胞因子（白细胞介素-1β、白细胞介素-6、白细胞介素-8和肿瘤坏死因子-α）对体外脂多糖（LPS）刺激反应的方法缺乏同时检测细胞内mRNA和蛋白质的能力。本程序利用新方法和仪器，在LPS刺激1、3和6小时后，同时测量CD14(+)单核细胞内的肿瘤坏死因子-α mRNA和蛋白质。通过评估多个时间点，我们能够辨别LPS刺激如何影响肿瘤坏死因子-α mRNA与蛋白质之间的时间关系。通过使用基于图像的流式细胞术，可以将mRNA和蛋白质信号共定位，以确定启动蛋白质翻译所需的孵育时间。