Fei Chenjie, Lillico Dustin M E, Hall Brian, Rieger Aja M, Stafford James L
Department of Biological Sciences, University of Alberta, Alberta, Canada.
EMD Millipore, Amnis, Seattle, Washington, USA.
Cytometry A. 2017 Apr;91(4):372-381. doi: 10.1002/cyto.a.23050. Epub 2017 Jan 12.
Innate immune cell-mediated recognition, capture, and engulfment of large particulate targets such as bacteria is known as phagocytosis. This highly dynamic cellular process involves a series of steps including receptor-mediated target binding, phagocytic cup formation, pseudopod extension, and phagosome closure, which depend on distinct actin polymerization events. Using flow cytometry, precise determination of target locations relative to cell membranes (i.e., surface-bound vs. fully engulfed/internalized) during the phagocytic process is difficult to quantify. Here, we describe the application of new analysis features within the IDEAS® software to distinguish internalized and surface-bound particles on individual cells with a high degree of accuracy and reproducibility. Through the use of connected component masks, the accurate discrimination of surface-bound beads versus those internalized is clearly demonstrated. In addition, we were able to further analyze the ratio of beads that had been surface-bound or internalized within individual cells. This novel method of analyzing the phagocytic process provides more accurate determination of target-cell interactions that will assist in examination of the signalling events that occur during the various stages of phagocytosis. © 2017 International Society for Advancement of Cytometry.
天然免疫细胞介导的对大型颗粒靶标（如细菌）的识别、捕获和吞噬被称为吞噬作用。这个高度动态的细胞过程涉及一系列步骤，包括受体介导的靶标结合、吞噬杯形成、伪足延伸和吞噬体闭合，这些步骤依赖于不同的肌动蛋白聚合事件。使用流式细胞术，在吞噬过程中精确确定靶标相对于细胞膜的位置（即表面结合与完全吞噬/内化）很难进行量化。在这里，我们描述了IDEAS®软件中新分析功能的应用，以高度准确和可重复的方式区分单个细胞上内化和表面结合的颗粒。通过使用连通组件掩码，清楚地展示了对表面结合的珠子与内化珠子的准确区分。此外，我们能够进一步分析单个细胞内表面结合或内化的珠子的比例。这种分析吞噬过程的新方法提供了对靶标 - 细胞相互作用更准确的测定，这将有助于检查吞噬作用各个阶段发生的信号事件。© 2017国际细胞计量学促进协会。
