CM enhances differentiation of EB into CD31-positive cells. Plated EB were treated either with (A,C) control medium or the supernatant of macrophages, which were prestimulated for 16 hours with the supernatant of apoptotic Jurkat cells and then incubated for 5 hours with fresh medium (CMPM). CMPM was derived from RAW264.7 (B,D), primary mouse wt macrophages (E), or primary mouse HIF-1α−/− macrophages (F), and added to EB for 24 hours. The appearance of CD31-positive cells was monitored by immunofluorescence staining (red). DAPI is stained in blue. Scale bar represents 200 μm (A-B) and 100 μm (C-F). Results are representative for 3 individual experiments. The dotted line indicates the border between the multilayer body and the surrounding monolayer rim of the EB. Original magnifications, ×10 (A-B) and ×40 (C-E). (G) Mean CD31 fluorescence intensity in the monolayer rim of EB was measured, and the ratio of CD31 versus DAPI under control conditions was set to 1. Data are the mean ± SD (n ≥ 3). *Significant alterations compared with controls (or otherwise as indicated).
