Lamb Evan R, Glomski Ian J, Harper Taylor A, Solga Michael D, Criss Alison K
Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA, USA.
Flow Cytometry Core Facility, University of Virginia, Charlottesville, VA, USA.
bioRxiv. 2024 Dec 5:2024.12.01.626241. doi: 10.1101/2024.12.01.626241.
Polymorphonuclear lymphocytes (PMNs) are terminally differentiated phagocytes with pivotal roles in infection, inflammation, tissue injury, and resolution. PMNs can display a breadth of responses to diverse endogenous and exogenous stimuli, making understanding of these innate immune responders vital yet challenging to achieve. Here, we report a 22-color spectral flow cytometry panel to profile primary human PMNs on population and single cell levels for surface marker expression of activation, degranulation, phagocytosis, migration, chemotaxis, and interaction with fluorescently labeled cargo. We demonstrate the surface protein response of PMNs to phorbol ester stimulation compared to untreated controls in an adherent PMN model with additional analysis of intra- and inter-subject variability. PMNs challenged with the Gram-negative bacterial pathogen revealed infectious dose-dependent changes in surface marker expression in bulk, population-level analysis. Imaging flow cytometry complemented spectral cytometry, demonstrating that fluorescence signal from labeled bacteria corresponded with bacterial burden on a per-cell basis. Spectral flow cytometry subsequently identified surface markers which varied with direct PMN-bacterium association as well as those which varied in the presence of bacteria but without phagocytosis. This spectral panel protocol highlights best practices for efficient customization and is compatible with downstream approaches such as spectral cell sorting and single-cell RNA-sequencing for applicability to diverse research questions in the field of PMN biology.
多形核淋巴细胞（PMNs）是终末分化的吞噬细胞，在感染、炎症、组织损伤和恢复过程中发挥着关键作用。PMNs 能够对多种内源性和外源性刺激表现出广泛的反应，这使得了解这些先天性免疫反应细胞至关重要，但也极具挑战性。在此，我们报告了一种 22 色光谱流式细胞术检测方法，用于在群体和单细胞水平上分析原代人 PMNs 的激活、脱颗粒、吞噬作用、迁移、趋化作用以及与荧光标记物质相互作用的表面标志物表达情况。在贴壁 PMN 模型中，我们展示了与未处理对照相比，PMNs 对佛波酯刺激的表面蛋白反应，并对个体内和个体间的变异性进行了额外分析。用革兰氏阴性细菌病原体攻击 PMNs，在大量群体水平分析中揭示了表面标志物表达的感染剂量依赖性变化。成像流式细胞术补充了光谱细胞术，证明了来自标记细菌的荧光信号与单个细胞上的细菌负荷相对应。光谱流式细胞术随后鉴定出了随 PMN 与细菌直接结合而变化的表面标志物，以及在有细菌存在但无吞噬作用时发生变化的表面标志物。这种光谱检测方法突出了高效定制的最佳实践，并且与下游方法如光谱细胞分选和单细胞 RNA 测序兼容，适用于 PMN 生物学领域的各种研究问题。
