Clone tdm29

Cell adhesion assays were performed as previously described [30] (link). 96-well microplates were coated with a solution of 10 µg/ml type 1 collagen from calf skin (Sigma-Aldrich; MO, USA) in PBS, or PBS-1% BSA. For each well, 2.5×10⁴ cells were seeded and incubated at 37°C for 20 min. In selected experiments, cells were previously incubated with function-blocking mAbs against integrin subunits α2 (clone P1E6, Chemicon; 1/50 in PBS), α3 (clone P1B5, Calbiochem, CA, USA; 1/50), α5 (clone P1D6, Chemicon; 1/50) or β1 (clone TDM29, Chemicon; 1/5) for 30 min at 4°C. After three washes, adherent cells were estimated with the MTT method (Sigma). Three independent experiments were performed in quadruplicate. Results were expressed as the mean ± standard deviation (SD) of values of specific binding to type 1 collagen (OD 570 nm of cells bound – OD 570 nm of cells bound to PBS-1% BSA).

Detection of integrin subunits and E-cadherin was performed by indirect immunofluorescence. Cells (5×10⁵) were incubated at 4°C for 30 min in the presence or absence of monoclonal antibodies (mAb) against integrin subunits β1 (clone TDM29, Chemicon, CA, USA; diluted 1/10), α2 (clone P1E6, Chemicon; 1/200) or mAb against human E-cadherin (clone HECD-1, Zymed Labs CA, USA; 1/50). After a wash, cells were incubated with the secondary antibody Alexa Fluor 488 goat anti-mouse IgG (Invitrogen Life Technologies, MD, USA). Antibodies were diluted in Phosphate Buffered Saline (PBS) containing 1% Bovine Serum Albumin (BSA). Mean Fluorescence Intensity (MFI) was calculated as the quotient between the positive and negative GeoMean for each cell line. For each sample three independent assays were performed.