Figure 5

Nuclear morphologic analysis of CD16+ cell populations in adipose tissue confirms flow cytometric phenotypic analysis. Imaging flow cytometry of adipose tissue immune cells was performed as in Figure 3. After exclusion of non-nucleated cells and Hoechst saturating the camera, poorly focused cells, and debris/doublets, CD3-CD14- cells were gated on CD16+ cells. CD16+ cells (~30 cells for each group) were manually selected (Tagged) based on their nuclear morphology to distinguish mononuclear (Circular, panel A) from polymorphonuclear (Polymorph, panel B) cells. The Feature Finder wizard in IDEAS software was used to identify similar cells in the total CD16+ source population (Automated, panels C, E) and to exclude debris and unfocused cells (Automated, panel D). Circularity and Bright Detail Intensity of the Hoechst imagery were the 2 characteristics that best distinguished the manually selected Polymorph and Circular cells. A plot of these 2 parameters for the Tagged and Automated CD16+ populations is depicted. BF = brightfield microscopy, Hoec = Hoechst nuclear staining. CD16 was plotted against SSC to compare Circular and Polymorph populations. Positive gating for each fluorochrome parameter was established using FMO controls.