Schappe Michael S, Desai Bimal N
Pharmacology Department, University of Virginia, Charlottesville, VA, USA.
Carter Immunology Center, University of Virginia, Charlottesville, VA, USA.
Bio Protoc. 2018 Jul 20;8(14). doi: 10.21769/BioProtoc.2926.
After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways-one from the plasma membrane and the other from the signaling endosomes (Kagan 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe 2018). The assay relies on stimulating the cells with LPS and measuring the cell surface levels of TLR4 (or CD14) at various time points using flow cytometry. Although we detail the method specifically for TLR4 and CD14 from murine bone marrow-derived macrophages, it can be readily adapted to evaluate receptor endocytosis in a variety of other signaling contexts.
识别细胞外细菌脂多糖（LPS）后，Toll样受体4（TLR4）-CD14信号复合物启动两条不同的信号通路——一条来自质膜，另一条来自信号内体（卡根，2008年）。因此，要了解TLR4信号转导的早期阶段，需要一种强大且定量的方法来测量LPS触发的TLR4和CD14受体内吞作用，这是LPS检测的最早事件之一。在此，我们描述了一种基于流式细胞术的方法，我们最近用该方法研究了离子通道TRPM7在TLR4内吞作用中的作用（沙佩，2018年）。该检测方法依赖于用LPS刺激细胞，并使用流式细胞术在不同时间点测量TLR4（或CD14）的细胞表面水平。尽管我们详细介绍了该方法在来自小鼠骨髓源性巨噬细胞的TLR4和CD14上的具体应用，但它可以很容易地适用于评估各种其他信号背景下的受体内吞作用。
