Flow cytometric analysis of novel surface markers identified by microarray. After doublet and dead cell exclusion with 7-aminoactinomycin D, gating on the 3 monocyte subsets for both specific and background staining was done based on CD14 and CD16 expression as shown in Figure 1A. Specific stainings are represented by blue tinted histograms, whereas FMO isotype controls are represented by histograms with red dotted lines. Surface markers were categorized according to the monocyte subset showing highest expression in microarray: (A) classical, (B) intermediate, and (C) nonclassical. Numbers in each histogram represent MFI of specific staining minus isotype control. (D) The mean ± SD of the MFI of the specific staining minus the MFI of its FMO isotype control for the surface antigens. n represents the number of persons on which the results for each surface marker is based. Statistical calculations of significance were performed using ANOVA followed by the post-hoc Tukey test for significant differences between any 2 monocyte subsets: *P < .05, **P < .01, ***P < .001. (E) The expression of representative markers was tracked based on increasing CD16 and decreasing CD14 expression. The intermediate monocyte subset was further subdivided based on increasing CD16 expression using R2, R3, and R4 gates. The nonclassical monocyte subset was further subdivided based on decreasing CD14 expression using R5, R6, and R7 gates. The classical subset was identified by R1 gate. Colored plots representing histograms from R1 to R7 were created using FlowJo software.