Monocyte subsets in PBMCs were explored. (A) PBMCs from a representative healthy blood donor were labeled with anti-CD45, -CD24, -CD14, -CD16, -CD56, -CD115, -CD62L, -CD64, -CCR2, and -CX3CR1 antibodies. Monocytes were identified using an exclusion gating strategy (described in supplemental Figure 1), and subsets were separated on CD14 and CD16 expression. The percentage of each subset is indicated. (B) May-Grünwald-Giemsa staining of sorted MO1s, MO2s, MO3s, and DN (remaining double-negative CD14⁻/CD16⁻) cells. (C) Multiparametric analysis of single cells monitored with 10 surface markers (supplemental Table 1) using the SPADE algorithm, which organizes cells in a hierarchy of related phenotypes. Flow cytometry data from 19 healthy donor PBMCs were gated on morphology, then on CD45⁺/SSC intermediate, and used to construct the SPADE tree that automatically separates, on the basis of the hierarchy of related phenotypes, MO1s (CD14⁺/CD16⁻), MO2s (CD14⁺/CD16⁺), MO3s (CD14^low/CD16⁺), natural killer cells (NK; CD56⁺), B lymphocytes (CD24⁺), and residual granulocytes (Gran; CD24⁺/CD16⁺). The percentage of each subset in the monocyte population is indicated. Circles indicate the size of cell populations, and colors are based on CD14 expression. (D) Color representation of CD16, CX3CR1, and CCR2 expression in the monocyte subsets delineated in the SPADE tree.