Fig. 3.

Aged peritoneal macrophages have abnormal oxysterol content. (a-c) Aged peritoneal macrophages contained significantly more intracellular 4β-hydroxycholesterol (4β-HC) and 7-ketocholesterol (7-KC) than their young counterparts at baseline and after treatment with 25 or 50 μg/ml oxidized LDL (oxLDL) (N = 5/group; 2-way ANOVA) and significantly more intracellular cholestane-3β,5α,6β-triol (C-triol) after treatment with 50 μg/ml oxLDL (N = 5/group; 2-way ANOVA with Bonferroni post-hoc test). (d-f) Although some comparisons were statistically significant due to low within-group variance, the supernatants of young and aged peritoneal macrophages contained qualitatively similar levels of 4β-HC, 7-KC, and C-triol both at baseline and after treatment with oxLDL (N = 5/group; 2-way ANOVA with Bonferroni post-hoc test). (g) Representative flow cytometry plot from young and aged peritoneal macrophages showing gating on macrophage markers CD64 and F4/80. (h-i) Young and aged peritoneal macrophages exhibited similar CD36 surface expression. Isotype staining (iso) was identical between groups (N = 5/group; 2-tailed, unpaired t-test). Open circles depict individual data points; horizontal lines depict mean ± SEM (a-f, i) (*** P < .001).