Immulon 2hb

Cell adhesion assays were performed in 96-well, flat-bottom microtiter plates (Immulon 2HB, Dynex Technologies, USA). The wells were coated overnight at 4°C with 50 μl of various amounts of the Fbln7-C recombinant protein. For peptide coating, various amounts of peptides in 50 μl of Milli-Q water were added to the wells and dried overnight at room temperature. The adhesion assay was performed as previously described ^{9 (link)}. Briefly, 3 × 10⁴ cells per well were plated and incubated for 2 hours at 37°C in a humidified atmosphere of 5% CO₂. Attached cells were fixed and stained for 10 min with 0.2% crystal violet in 20% methanol. The cells were dissolved in 1% sodium dodecyl sulfate (SDS), and the absorbance at 570 nm was measured. For the inhibition of cell attachment with heparin (Sigma-Aldrich, USA), EDTA (Sigma-Aldrich, USA) and integrin β1-blocking antibody (EMD Millipore, USA), HUVECs were first incubated for 20 min at 37°C in the presence of either 10 μg/ml heparin, 5mM EDTA, or 10 μg/ml integrin β1-blocking antibody (clone 6S6, Millipore). Data are expressed as mean ± SD. Statistical analysis were performed using the t test analysis.