Adhesion and spreading of monocytes on amphoterin and extracellular matrix proteins. (A-C) Peripheral blood leukocytes were kept on coverslips coated with vitronectin (A), fibronectin (B), or recAtn (C) for 1 hour in serum-free medium. The coverslips were washed, fixed with 2% paraformaldehyde, and immunostained with anti-CD14 and TRITC-labeled antimouse immunoglobulins. Scale bar (A-C) 10 μm. (D) Measurement of amphoterin and fibronectin adherent monocyte areas. Monocytes were adhered to recAtn or fibronectin-coated plastic for 1 hour, and adherent cells were fixed. Digital microscopy pictures were taken, and cell areas were measured. Mean of areas was calculated. Amphoterin-adherent cells spread strongly compared with fibronectin-adherent cells, and spreading was inhibited with 100 g/mL sRAGE but not with sAMIGO. Error bars represent ± SD (n = 3). (E) Peripheral blood mononuclear cells in serum-free medium were incubated in microwells coated with recAtn, fibronectin (Fn), or albumin (BSA) for 1 hour, and the bound CD14⁺ cells were detected with anti-CD14 and ELISA. (F-G) Peripheral blood leukocytes were incubated on recAtn or Fn-coated coverslips for 1 hour in serum-free medium, and the bound cells were immunostained with anti-CD14 and TRITC-labeled second antibody. The number of CD14⁺ cells (F) and their proportion of total cells (G) were determined by fluorescence and phase-contrast microscopy. The mean and SEM of 3 different experiments (E) or 12 fields of 2 different experiments (F-G) are shown.