Analysis and isolation of circulating monocytes. Monocyte subsets were gated on the basis of (A) forward scatter (FSC) and side scatter (SSC) and (B) singlets and CD14/CD16 expression. Resulting monocyte (C) percentages and (D) numbers showed CMs to be the most common circulating monocyte subset, followed by NCMs and IMs. (E) Fluorescence-activated cell sorter analysis of 200 sorted cells from all 3 monocyte subsets typically revealed a purity >99% and good separation between the populations. Note that the inclusion of pan-monocyte markers CD86 and major histocompatibility complex II in our gating strategy (either with or without the inclusion of cells in the lymphocyte gate) resulted in a slightly higher number of CMs and IMs recovered (1% and 2% higher counts on average, respectively) and a lower number of recovered NCMs (3%). However, use of these cell numbers did not result in significant differences in parameter estimates. (F) Cytospin preparations stained with May-Grünwald-Giemsa showed an increasingly mature phenotype from CMs to NCMs as characterized by a more neutrophilic cytoplasm and increasingly dendritic appearance. The objective was a 100× oil immersion lens, numerical aperture was 1.30, and scale bars = 10 μm. (G) DNA ²H-enrichments after in vivo pulse-labeling with 6,6-²H₂-glucose were determined by gas chromatography-mass spectrometry and plotted after normalization for plasma enrichment and intracellular dilution of label (supplemental Methods). Results are based on a total of 249 measurements from 14 individuals; circles indicate medians, and bars represent interquartile ranges. (A-B,E-F) Representative results from 1 experiment. (C-D) Circles represent the medians of 6 measurements for each volunteer, and lines indicate medians for the 14 volunteers.