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+ CX3CR1^GFP+, and CD11b+ CX3CR1^GFP-), 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).