Purification of human monocyte subsets and microarray analysis. (A) The classical (CD14⁺⁺CD16⁻), intermediate (CD14⁺⁺CD16⁺), and nonclassical monocyte (CD14⁺CD16⁺⁺) subsets were purified from 4 healthy donors using FACS. RNA was obtained and gene expression profiling was performed using Illumina Human-6 Version 2 BeadChip arrays. Duplicate samples for each donor were analyzed. (B) Gene expression profiles were subjected to hierarchical clustering using city block distance, average linkage (C) and principle component analysis. Shapes represent expression profiles of the different donors in duplicate. The different monocyte subsets are circled and are represented by different colors. Green, purple, and red represent the classical, intermediate, and nonclassical subset, respectively. (D) Number of comparisons with ≥ 1.5-fold change within the 1554 differentially expressed genes. The length of the lines between 2 monocyte subsets is proportional to the number of such comparisons.