Leeson Hannah C, Chan-Ling Tailoi, Lovelace Michael D, Brownlie Jeremy D, Weible Michael W, Gu Ben J
Griffith Institute for Drug Discovery, Griffith University; Australian Institute for Bioengineering and Nanotechnology, University of Queensland.
Discipline of Anatomy and Histology, School of Medical Science, University of Sydney; Bosch Institute, University of Sydney.
J Vis Exp. 2019 Apr 3(146). doi: 10.3791/59313.
Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.
活细胞流式细胞术在细胞生物学家中越来越多地被用于量化活细胞培养中的生物学过程。本方案描述了一种扩展活细胞流式细胞术的方法，用于实时分析P2X7受体激活的多种功能。使用安装在流式细胞仪上的时间模块，可以在给定时间段内评估活细胞功能并进行绘图，以探索钙内流、跨膜孔形成和吞噬作用的动力学。这种简单的方法具有优势，因为P2X7受体的所有三种典型功能都可以使用一台仪器进行评估，并且随着时间绘制的收集数据提供了关于整个活细胞群体的信息，而不是通常使用技术上具有挑战性的膜片钳方法获得的单细胞记录。钙内流实验使用钙指示剂染料，而P2X7孔形成测定依赖于在高激动剂浓度下允许溴化乙锭穿过形成的跨膜孔。黄绿（YG）乳胶珠用于测量吞噬作用。应用特定的激动剂和拮抗剂来研究P2X7受体活性的影响。单独地，这些方法可以进行修改，以提供关于任何数量的钙通道、嘌呤能和清道夫受体的定量数据。综上所述，它们突出了实时活细胞流式细胞术的使用是一种快速、经济高效、可重复且可量化的研究P2X7受体功能的方法。
