Role of endoglin in static and flow cell adhesion assays. (A,B,D,E) Static cell adhesion. Plates were coated with BSA, human endoglin (Eng), mouse endoglin (mEng; Fc fusion construct), CXCL12 (CXC) or human IgG (used as control of the Fc part of mEng fusion protein), as indicated. Human Nalm6 cells (A), B lymphocytes (B) and K562 cells (E), as well as murine SP2 cells (D) were labeled with CFSE, loaded in the wells (1 × 105 cells/well) and incubated with or without soluble endoglin or the RGD tripeptide, as indicated for 1h at 37°C. Bound cells were lysed and quantification was carried out using a fluorescent analyzer. Results are shown as percentage respect to total cell input (100%). Each assay was performed in triplicate and the SEM is indicated. (*P < .01; **P < .005). (C) Flow adhesion assay. Coated plates were incorporated as the lower wall of a flow chamber and mounted on an inverted microscope equipped with a monochromatic camera. Nalm6 cells were infused and allowed to bind to the substrate before flow rate was adjusted to 1 dyne/cm2 and then increased every 30 seconds. The percentages of cells that remained bound are represented in the vertical axis. A representative video of Nalm6 cells bound to Eng/CXCL12 under increasing flow rates is available online (see supplemental Video 1).
