Activation of Eph receptor by its ligand increases adhesion to fibronectin but not to PLL. (A) Ninety-six-well plates were coated with serial dilution of fibronectin or poly-l-lysine (PLL) (1-20μg/mL) overnight at 4°C. Nonspecific binding sites were blocked at rt for 1 hour using 1% BSA/PBS. Day-11 CD34+-derived DCs (1 × 105 cells in 100 μL serum-free medium) were plated in the presence of ephrin-A3-Fc (10 μg/mL; ▪) or control immunoglobulin (10 μg/mL; ▵) and centrifuged at 1200 rpm for 3 minutes. After washing with PBS and fixation with 0.5% paraformaldehyde/0.5% glutaraldehyde at rt for 30 minutes, adherent cells were stained with 0.5% crystal violet in 20% methanol at rt for 10 minutes. The cells were washed 3 times with water and were extracted with 50% ethanol/50 mM sodium citrate, pH 4.5. Values represent mean ± SD A570 absorbances from triplicate wells. Results are representative of 3 independent experiments. (B-D) Ninety-six-well plates were coated with 5 μg/mL fibronectin (B), PBS (C), or PLL (D) overnight at 4°C. Nonspecific binding sites were blocked at rt for 1 hour using 1% BSA/PBS. Day-11 CD34+-derived DCs (1 × 105 cells in 100 μL serum-free medium) were plated in the presence of PBS (control; light gray bars), Fas-Fc (white bars), ephrin-A3-Fc (hatched bars), ephrin-B3-Fc (dark gray bars), or ephrin-A4-Fc (10 μg/mL; black bars) and were centrifuged at 1200 rpm for 3 minutes. After washing and fixation, adherent cells were stained with crystal violet. Values represent mean ± SD A570 absorbances from triplicate wells. The results comparing the different ephrin-Fc chimera are representative of 3 independent experiments, and overall the results with ephrin-A3-Fc are representative of 13 experiments. O.D. = optical density.
