FACS analysis of ECs detected in a patient affected by Systemic Sclerosis. Panel A: Before iloprost infusion; Panel B: After iloprost infusion. Sequential four-color gating strategy. In cytogram (a) which displays all events, a rectangular region (R1) is drawn to exclude dead cells from analysis (7-AAD positive-cells). In cytogram (b), a polygonal region (R2) is drawn to define lymphocytes on the basis of the morphological parameter Side Scatter (SSC) and of CD45 expression. An additional region (R3), which includes all CD45 positive events, is depicted to derive CECs and EPCs enumeration. In cytogram (c), R4 is defined as FSC (Forward Scatter)/SSC gate on lymphocytes set on FSC left-hand border and include intermediate region between lymphocytes and monocytes. In cytogram (d) are included all events which meet morphological criteria of R4. R5 and R6 include respectively CECs and EPCs which are shown negative for dump channel markers (CD3/CD16/CD19/CD33) in cytogram (e). CECs and EPCs show a different staining with CD146/CD31/CD133/CD34 (ECs). Cytogram (f) shows the morphological characteristics of cells in R5 and R6 (CECs and EPCs respectively).