成像流式细胞仪在细胞吞噬研究中的应用
首发时间:2015-12-14
摘要:吞噬在天然免疫和发育等多种基础生物学以及疾病过程中发挥重要作用。目前分析吞噬的方法,主要限于流式细胞仪和手动成像分析,提供的信息有限,无法实现高通量的快速筛选和吞噬定位。本文应用高速成像流式细胞仪这一以成像为基础的自动化分析系统,定量分析吞噬过程的结合和内化。本文将PE-F4/80标记的腹腔巨噬细胞和NBD-PC标记的磷酯酰丝氨酸(Phosphatidylserine, PS)修饰微珠共孵育后,首先运用流式细胞仪分析巨噬细胞吞噬水平,将PE和NBD双阳性信号的细胞定义为吞噬细胞(Phagocytic cells)。结果可见共孵育后,细胞松弛素D(Cytochalasin D)处理后可抑制巨噬细胞吞噬,吞噬率从63.83±6.30%(正常组)下降至32.66±4.79%(Cytochalasin D 处理组),提示一部分微珠仅和巨噬细胞结合,而并未内化吞入巨噬细胞胞内。进一步运用成像流式细胞仪检测吞噬水平, Internalization 模块统计细胞内的荧光(代表微珠的绿色)在整个细胞荧光(代表巨噬细胞的橙色)中的百分比,该数值代表吞噬过程中的内化作用;而细胞边缘的荧光在整个细胞荧光中的百分比则代表吞噬过程中的结合作用。结果显示双阳性信号的细胞中,约64%为吞噬了微珠的细胞,另有约36%为结合微珠的细胞。因此,运用成像流式细胞仪可以准确定量吞噬中真正发生内化的细胞,区别开处于结合状态的细胞,为研究者们提供了研究吞噬作用的高通量方法。
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Application of image-based flow cytometry in cell phagocytosis
Abstract:Phagocytosis is an important cellular process in innate immunity, development, and disease. Current methods for analyzing phagocytosis are largely limited to flow cytometry and manual image-based assays, providing limited information. Here, we use image flow cytometry, an image-based and automated assay to rapidly quantitate binding and internalization stages during phagocytosis. PE-F4/80 labeled peritoneal macrophages were incubated with NBD-labeled PS beads, flow cytometry was first performed to analyze phagocytosis, double positive cells were defined as phagocytic cells. It showed that the phagocytic percentage decreased from 63.83±6.30 % in ctrl to 32.66±4.79 % in Cytochalasin D treated (P<0.05), indicating that there were about 30% beads were bound to but not internalized into macrophage. The phagocytic level was also statisticaled by the Internalization wizard in image flow cytometry. The beads that overlapped with two peripheral pixels of phagocytes were considered as bound but not internalized; the rest was considered as internalized. It showed that the binding and internalization percentage were 64%, 36% respectively. Image flow cytometry offers the ability to automatically distinguish large amounts of image data into the binding and internalization within minutes, clearly demonstrating its potential value in investigating phagocytosis.
Keywords: cell biology Phagocytosis Intrenalization binding Image stream flow cytometry
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