Figure 4

Adhesion of H9 cells on an hE-cad-Fc coated surface. (A) Number of adherent H9 cells was determined for hE-cad-Fc-coated and Matrigel-coated dishes following incubation for 3-hours in conditioned media (black) or mTeSR1 (white). The data are presented as means ± SD of 3 independent experiments. *: p < 0.01, **: p < 0.05. (B, C) E-cadherin was identified by immunoblot (B) or by FACS (C) after collecting cells by scraping (NT), Accutase (Acc) or enzyme free Cell Dissociation Buffer (CDB). When cells were incubated for <5 mins in Accutase (short) (C) a subset of cells retained cell surface E-cadherin, which facilitated plating (A). (D) Adhesion of H9 cells onto the hE-cad-Fc-coated surface was restored by CDB treatment. H9 cells cultured on hE-cad-Fc- or Matrigel-coated surface were treated with Accutase (Acc) or CDB. The adhesion of cells onto hE-cad-Fc (open bar) or Matrigel (closed bar) was analyzed by WST-1 assay. The data are means ± SD of 3 independent experiments. *: p < 0.01, N.S.: non significant. (E) The concentration of immobilized hE-cad-Fc was analyzed by HRP-labelled hE-cad-Fc, and fitted to a Langmuir isotherm curve. The data are means ± SD of 3 independent experiments. (F) H9 cells were treated with CDB and seeded onto the indicated concentration of hE-cad-Fc-coated surface in mTeSR1 media. The number of adhered cells was counted by WST-1 reagent and compared with the cell number on 20 μg/ml of hE-cad-Fc. The data are means ± SEM of 6 independent experiments.