Fig. 5

Flow cytometric detection and enumeration of CEC from peripheral blood. CEC frequency was assessed via a composite analysis, consisting of three separate flow cytometric analyses of a single blood sample after erythrocyte lysis. a-d: ori sample, not immuno-magnetically enriched; e-h: iso sample, immuno-magnetically enriched for CD34+ cells, antibody for CEC indicative CD146+ staining exchanged with isotype matched unspecific antibody; j-m: stain sample, immuno-magnetically enriched for CD34+ cells, with specific antibody for CEC indicative CD146+ staining. a, e, j: Dot plots for exclusion of debris and remaining erythrocytes (x-axis: forward scatter FCS; y-axis: sideward scatter, SSC) resulting in gate P1. b, f, k: Dot plots for exclusion of dead cells (x-axis: CD45; y-axis: propidium iodide, PI) gated in P1 resulting in gate P2 (P1/P2). c, g, l: Dot plots for exclusion of hematopoietic cells (x-axis: CD45; y-axis: CD34) gated in P2 resulting in gate P3 (P1/P2/P3). d, h, m: Dot plots for validation of CEC (x-axis: CD34; y-axis: CD146) gated in P3 resulting in gate P4 (P1/P2/P3/P4). The number displayed in the P4 gate represents the absolute number of detected CEC in the respective fraction of the blood sample (ori: 100 μl; iso: 5 ml; stain: 5 ml)