Caponegro Michael D, Thompson Kaitlyn Koenig, Tayyab Maryam, Tsirka Stella E
Molecular and Cellular Pharmacology Program, Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794-8651, USA.
Bio Protoc. 2020 Aug 5;10(15). doi: 10.21769/BioProtoc.3698.
Studying monocytic cells in isolated systems contributes significantly to the understanding of innate immune physiology. Functional assays produce read outs which can be used to measure responses to selected stimuli, such as pathogen exposure, antigen loading, and cytokine stimulation. Integration of these results with high quality models allows for the development of therapeutics which target these cell populations. Current methodologies to quantify phagocytic function of monocytic cells either measure phagocytic activity of individual cells (average number of beads or particles/cell), or a population outcome (% cells that contain phagocytosed material). Here we address technical challenges and shortcomings of these methods and present a protocol for collecting and analyzing data derived from a functional assay which measures phagocytic activity of macrophage and macrophage-like cells. We apply this method to two different experimental conditions, and compare to existing work flows. We also provide an online tool for users to upload and analyze data using this method.
在分离系统中研究单核细胞对理解固有免疫生理学有显著贡献。功能测定产生的读数可用于测量对选定刺激的反应，如病原体暴露、抗原负载和细胞因子刺激。将这些结果与高质量模型相结合有助于开发针对这些细胞群体的疗法。目前量化单核细胞吞噬功能的方法要么测量单个细胞的吞噬活性（珠子或颗粒/细胞的平均数），要么测量群体结果（含有吞噬物质的细胞百分比）。在这里，我们阐述了这些方法的技术挑战和缺点，并提出了一种用于收集和分析来自测量巨噬细胞和巨噬样细胞吞噬活性的功能测定数据的方案。我们将此方法应用于两种不同的实验条件，并与现有工作流程进行比较。我们还提供了一个在线工具，供用户使用此方法上传和分析数据。
