Slarve Matthew, Nielsen Travis, Yu Dongran, VanOrman Jordyn, Luna Brian, Spellberg Brad
Molecular Microbiology and Immunology Department, University of Southern California, Los Angeles California, United States of America.
Los Angeles General Medical Center, Los Angeles, California, United States of America.
PLoS One. 2025 Sep 9;20(9):e0331445. doi: 10.1371/journal.pone.0331445. eCollection 2025.
Assessing the phagocytosis of microbes by macrophages is an important component of studies of novel immunotherapeutics, antimicrobial drugs, immune effectors, or any immunology related research. Here we define two protocols for measuring in vitro phagocytosis by RAW 246.7 cells - a photographic phagocytosis assay that allows optical measurement of bacterial cells inside of the RAW 246.7 cell by staining fixed cells and visually quantifying the bacteria that have been phagocytosed, as well as a killing assay, which measures the viable bacteria released from lysed cells following exposure and phagocytosis via colony forming unit analysis. These methods differ from previously available protocols for measuring phagocytosis, as they are more generalizable to a variety of microbes and experimental designs and use readily available cell lines and materials to produce robust results. We demonstrate the utility of our methods by showing how opsonization by our novel, therapeutic monoclonal antibodies drive phagocytosis and killing of the gram-negative bacteria, Acinetobacter baumannii.
评估巨噬细胞对微生物的吞噬作用是新型免疫疗法、抗菌药物、免疫效应器或任何免疫学相关研究的重要组成部分。在此，我们定义了两种用于测量RAW 246.7细胞体外吞噬作用的方案——一种摄影吞噬试验，通过对固定细胞进行染色并直观量化已被吞噬的细菌，从而对RAW 246.7细胞内的细菌细胞进行光学测量，以及一种杀伤试验，该试验通过菌落形成单位分析来测量暴露和吞噬后从裂解细胞中释放的活细菌。这些方法与以前可用的测量吞噬作用的方案不同，因为它们更适用于多种微生物和实验设计，并使用现成的细胞系和材料来产生可靠的结果。我们通过展示我们新型治疗性单克隆抗体的调理作用如何驱动对革兰氏阴性菌鲍曼不动杆菌的吞噬和杀伤，证明了我们方法的实用性。
