Figure 3

Confirmation of immune cell populations in psoriatic adipose tissue using imaging flow cytometry. Morphologic and staining characteristics of various cell populations delineated by conventional flow cytometry (Figures 1 and 2) were confirmed in psoriatic adipose tissue using imaging flow cytometry. Cells were gated for positive nuclear staining that did not saturate the camera. Out-of-focus cells were excluded using the gradient RMS function of the brightfield microscopy field. Doublets and debris were eliminated by plotting cell brightfield area against aspect ratio. To determine nuclear morphology population characteristics, manually selected mononuclear or polymorphonuclear cells were identified and hand tagged populations were created. The Feature Finder wizard in IDEAS software was then used to distinguish cells with similar morphologic characteristics in the source population using the circularity and bright detail intensity features on the Hoechst imagery. Three populations of cells were identified: CD3+CD16- T cells (panel D), CD3-CD16+ cells, and CD3-CD16- cells. CD3-CD16- cells were then gated on CD14+ cells (macrophages), followed by sub-gating based on CD206 and HLADRII (DRII) staining (panels A-C). Positive gating for each fluorochrome parameter was established using FMO controls. Percentages of cells in each gate are presented as percentages of nucleated, focused, single cells.