Macrophage ablation via PLX3397 treatment did not affect peripheral immune cells in the spleen (Experiment 2) (Manuscript: Fig. S4-3)

Mice received either control chow or PLX3397-formulated chow for seven days to facilitate macrophage depletion. Following initiation of cisplatin administration, mice received daily PLX3397 treatment via oral gavage, which continued until euthanasia, ensuring sustained macrophage ablation (Experiment 2). Spleens harvested after endpoint auditory test were disrupted to obtain single cell suspension. Red blood cell (RBC) lysis was performed. Spleen single cell suspensions were pre-treated with anti-mouse CD16/CD32 antibody to block Fc receptors followed by surface staining with antibodies. Cells were then washed and incubated in Fixable Viability dye eFluor™450. Cell surface markers were used to identify specific populations of immune cells, including macrophages (CD11b+, CD11b+ CX3CR1GFP+, and CD11b+ CX3CR1GFP-), neutrophils (CD11b+ F4/80- Ly6G+), NK cells (CD3e- NK1.1+), T cells (CD11b- CD3e+), and B cells (CD11b- CD19+). The data suggest that macrophage ablation via PLX3397 treatment does not affect peripheral immune cells in the spleen (Experiment 2).

为实现巨噬细胞耗竭，小鼠分别喂食对照饲料或含PLX3397的配方饲料，持续7天。在启动顺铂（cisplatin）给药后，小鼠每日通过口服灌胃（oral gavage）给予PLX3397，该给药持续至安乐处死，以维持巨噬细胞的持续清除（实验2）。终点听觉测试结束后，采集小鼠脾脏并制备为单细胞悬液（single cell suspension），随后进行红细胞（red blood cell, RBC）裂解处理。脾脏单细胞悬液先经抗小鼠CD16/CD32抗体（anti-mouse CD16/CD32 antibody）预处理以阻断Fc受体（Fc receptor），再用相关抗体进行表面染色。细胞经洗涤后，于可固定活性染料eFluor™450（Fixable Viability dye eFluor™450）中孵育。通过细胞表面标志物鉴定免疫细胞的特定亚群，包括巨噬细胞（macrophages，表型为CD11b+、CD11b+ CX3CR1GFP+及CD11b+ CX3CR1GFP-）、中性粒细胞（neutrophils，表型为CD11b+ F4/80- Ly6G+）、自然杀伤细胞（NK cells，表型为CD3e- NK1.1+）、T细胞（T cells，表型为CD11b- CD3e+）及B细胞（B cells，表型为CD11b- CD19+）。本实验数据表明，经PLX3397处理实现的巨噬细胞清除，并不会影响脾脏内的外周免疫细胞群（实验2）。