Figure 2.

Imaging flow cytometry analysis of monocyte subpopulations. PBMC (n = 5) were stained and analysed by imaging flow cytometry (ImageStream, Amnis). After exclusion of lymphocytes, selection of CD14-positive cells, and doublet exclusion, small and large monocytes were visualized. (a) Representative images of small and large monocytes. (b) Representative images of cells in the CD14⁺ doublet gate. (c) Shape analysis of all events in small and large monocyte gates and in doublet gate using the aspect ratio feature (IDEAS software) in a representative donor. The ratio between the minor and major axis of each event in monocyte gates was calculated and compared to the corresponding ratio of cells in the doublet gate. A vertical bar drawn at the nadir between singlet and doublet curves (Aspect Ratio Intensity around 0.7) served as threshold to quantify singlets and doublets in each population. (d) Quantification of doublets present in gates used to define small and large monocyte subpopulations, calculated as events located left of the threshold bar drawn in panel c (mean ± s.d.) and expressed as percent of cells in the gate; doublets represented less than 5% of the cells in large monocyte gates, a percentage similar to that of small monocyte gates. (e) Cell size was determined using Area Feature (IDEAS software) with a mask delimited by CD14 expression in small and large monocytes, (f) in small monocyte subpopulations, and (g) in large monocytes subpopulations from a representative donor. (h) quantification of cell sizes for each subpopulation in 5 donors; sm14⁺16^neg, sm14⁺16⁺, and sm14^dim16⁺ monocytes had a similar size distribution, and large monocyte subpopulations la14⁺16^neg and la14⁺16⁺ had also very similar sizes (mean ± SEM, **p ≤ 0.01; ****p ≤ 0.0001, one way ANOVA).