(A) Plasma bilirubin in Spta^sph/sph and Spta^wt/wt (WT) mice (n = 4 for both groups, n.m. = not measurable). (B) Flow cytometry density plots of liver cell suspensions stained for F4/80 and intracellular TER-119 in Spta^sph/sph mice (red) and Spta^wt/wt littermates (blue). The displayed cells were gated from live CD45⁺ leukocytes. Data are representative of 3 independent experiments. Percentage proportions of cell counts are displayed in the plot. (C) Brightfield and fluorescence images showing intracellular TER-119⁺ erythrocytes (yellow) in F4/80⁺ Kupffer cells (KCs) (purple) from Spta^wt/wt and Spta^sph/sph mice obtained by imaging flow cytometry. (D) Top panel: Microphotographs of liver tissues stained for F4/80. Bottom panel: High-magnification microphotographs of F4/80⁺ macrophages from Spta^wt/wt and Spta^sph/sph mice. Data representative of 3 mice in each group. (E) Single-cell RNA sequencing (scRNA-seq) data of nonparenchymal liver cell suspensions enriched for macrophages with F4/80 antibody–coated magnetic Dynabeads from Spta^sph/sph and Spta^wt/wt mice. t-SNE plots showing color-coded Gypa/Hba-a1 gene expression in Spta^wt/wt and Spta^sph/sph mice. Double-positive cells are highlighted by the blue dashed line. Each dot represents a single cell. (F) Data sets from the 2 mice shown in E are merged and represented in a single t-SNE plot, showing 17,127 cells colored by cell origin. (G) Same t-SNE as in F. After an unsupervised clustering analysis, cells sharing similar transcriptome profiles were grouped by color. Seventeen clusters were identified. (H) Heatmap of the average expression of key marker genes for hepatocytes, endothelial cells, erythroblastic island macrophages (EBIs), early recruited macrophages, and KCs in each cluster. Data are standardized by row, Z scores are displayed by color code (red = high expression, blue = low expression). (I) Assigned cell populations defined in H. Cells are colored based on their identities.