Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Providence, RI, United States.
Warren Alpert Medical School, Brown University, Providence, RI, United States.
Front Immunol. 2023 Jan 23;14:1089111. doi: 10.3389/fimmu.2023.1089111. eCollection 2023.
Techniques for studying the clearance of bacterial infections are critical for advances in understanding disease states, immune cell effector functions, and novel antimicrobial therapeutics. Intracellular killing of by neutrophils can be monitored using a strain stably expressing GFP, a fluorophore that is quenched when exposed to the reactive oxygen species (ROS) present in the phagolysosome. Here, we expand upon this method by developing a bi-fluorescent killing assay for use Conjugating with a stable secondary fluorescent marker enables the separation of infected cell samples into three populations: cells that have not engaged in phagocytosis, cells that have engulfed and killed , and cells that have viable internalized . We identified ATTO647N-NHS Ester as a favorable dye conjugate for generating bi-fluorescent due to its stability over time and invariant signal within the neutrophil phagolysosome. To resolve the utility of ATTO647N/GFP bi-fluorescent , we evaluated neutrophil function in a murine model of chronic granulomatous disease (CGD) known to have impaired clearance of infection. Analysis of bronchoalveolar lavage (BAL) from animals subjected to pulmonary infection with bi-fluorescent demonstrated differences in neutrophil antimicrobial function consistent with the established phenotype of CGD.
研究细菌感染清除的技术对于深入了解疾病状态、免疫细胞效应功能和新型抗菌治疗方法至关重要。使用稳定表达 GFP 的 菌株可以监测中性粒细胞对 的细胞内杀伤作用，GFP 是一种荧光团，当暴露于吞噬体中的活性氧 (ROS) 时会被猝灭。在这里，我们通过开发一种用于 的双荧光 杀伤测定法对该方法进行了扩展，将 与稳定的二级荧光标记物缀合可以将感染细胞样品分离成三个群体：未参与吞噬作用的细胞、吞噬并杀死 的细胞以及含有存活内化 的细胞。我们确定 ATTO647N-NHS Ester 是一种有利的染料缀合物，因为它在中性粒细胞吞噬体中具有时间稳定性和不变的信号，适用于生成双荧光 。为了解决 ATTO647N/GFP 双荧光 的实用性，我们在慢性肉芽肿病 (CGD) 小鼠模型中评估了中性粒细胞功能，该模型已知清除 感染能力受损。对用双荧光 进行肺部感染的动物的支气管肺泡灌洗液 (BAL) 进行分析，结果显示中性粒细胞抗菌功能存在差异，与 CGD 的既定表型一致。
