Smirnov Asya, Solga Michael D, Lannigan Joanne, Criss Alison K
Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA.
Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA.
J Immunol Methods. 2015 Aug;423:60-9. doi: 10.1016/j.jim.2015.04.028. Epub 2015 May 9.
Recognition, binding, internalization, and elimination of pathogens and cell debris are important functions of professional as well as non-professional phagocytes. However, high-throughput methods for quantifying cell-associated particles and discriminating bound from internalized particles have been lacking. Here we describe a protocol for using imaging flow cytometry to quantify the attached and phagocytosed particles that are associated with a population of cells. Cells were exposed to fluorescent particles, fixed, and exposed to an antibody of a different fluorophore that recognizes the particles. The antibody is added without cell permeabilization, such that the antibody only binds extracellular particles. Cells with and without associated particles were identified by imaging flow cytometry. For each cell with associated particles, a spot count algorithm was employed to quantify the number of extracellular (double fluorescent) and intracellular (single fluorescent) particles per cell, from which the percent particle internalization was determined. The spot count algorithm was empirically validated by examining the fluorescence and phase contrast images acquired by the flow cytometer. We used this protocol to measure binding and internalization of the bacterium Neisseria gonorrhoeae by primary human neutrophils, using different bacterial variants and under different cellular conditions. The results acquired using imaging flow cytometry agreed with findings that were previously obtained using conventional immunofluorescence microscopy. This protocol provides a rapid, powerful method for measuring the association and internalization of any particle by any cell type.
识别、结合、内化以及清除病原体和细胞碎片是专职和非专职吞噬细胞的重要功能。然而，一直缺乏用于量化细胞相关颗粒并区分结合颗粒与内化颗粒的高通量方法。在此，我们描述了一种使用成像流式细胞术来量化与一群细胞相关的附着和吞噬颗粒的方案。将细胞暴露于荧光颗粒，固定后，再暴露于识别这些颗粒的不同荧光团的抗体。在不进行细胞通透处理的情况下添加抗体，使得抗体仅结合细胞外颗粒。通过成像流式细胞术识别有无相关颗粒的细胞。对于每个有相关颗粒的细胞，采用斑点计数算法来量化每个细胞的细胞外（双荧光）和细胞内（单荧光）颗粒的数量，由此确定颗粒内化百分比。通过检查流式细胞仪获取的荧光和相差图像，对斑点计数算法进行了经验验证。我们使用该方案，在不同细胞条件下，利用不同的细菌变体，测量了原代人中性粒细胞对淋病奈瑟菌的结合和内化。使用成像流式细胞术获得的结果与先前使用传统免疫荧光显微镜获得的结果一致。该方案提供了一种快速、强大的方法，用于测量任何细胞类型对任何颗粒的结合和内化。
