Single-cell sequencing and protein analysis revealed potential biomarkers for LPS + Ado and LPS + PGE₂human regulatory macrophages. Human monocytes were cultured in M-CSF for 7 days and then were left unstimulated or were stimulated with LPS, LPS + Ado, or LPS + PGE₂. (A) Violin plots are used to visualize the single-cell expression of secreted marker genes THBS1 and VEGFA. (B) Levels of the proteins THBS1 and VEGF in the supernatants of 24-hour stimulated M-CSF (circles) and GM-CSF (squares) macrophages were detected by ELISA (n = 7). *P ≤ .05; **P ≤ .01 relative to LPS stimulation alone; #P ≤ .05; ##P ≤ .01 between M-CSF and corresponding GM-CSF stimulation. (C) Violin plots are used to visualize the single-cell expression of surface-expressed marker genes CD300E and PLAUR. (D) Surface expression of CD300E (n = 5) and PLAUR (n = 3) on M-CSF (circles) and GM-CSF (triangles) macrophages stimulated for 24 hours or 8 hours was detected by flow cytometry and expressed as the median fluorescence intensity (MFI). *P ≤ .05; **P ≤ .01 relative to LPS stimulation alone; #P ≤ .05 between M-CSF and corresponding GM-CSF stimulation. Error bars represent SEM.