α β integrin mediated cell adhesion array combo kit

To characterize the integrin expression pattern of control and CD44 knockdown cells, an α/β Integrin-mediated Cell Adhesion Array Combo Kit (ECM532, EMD Millipore) was used, in which antibodies specific for a particular integrin subunit or heterodimer are immobilized onto microtiter wells. Cells were non-enzymatically harvested by incubation in 5 mM EDTA for 20 m, then incubated on microtiter plates for 2 h in serum-free medium. Non-adherent cells were gently washed off, and the number of cells was quantified with a colorimetric readout per manufacturer’s instructions. Each integrin subunit or heterodimer antibody was assayed in duplicate wells, and the experiment was repeated three times. The specificity of each antibody was verified, and published accounts of the antibody clones (20 (link), 21 (link)) used differed slightly from the kit description; these literature-validated antibody specificities are reported here.

The α/β-Integrin-Mediated Cell Adhesion Array Combo Kit (ECM532, Merck Millipore, Billerica, MA) was used according to the manufacturer’s instructions. Each well containing mouse anti-alpha or anti-beta integrin received 100μl containing 1.5x10⁵ cells, as did the BSA-coated negative control wells. The plate was incubated for 2hours at 37ºC in 5%CO₂ and washed with assay buffer. Cells were then stained with a Cell Stain Solution (provided in kit), incubated for 5min and washed with dH₂O. Extraction buffer (100μl) was added to each well and left to shake for 5-10min. Fluorescence was measured in Microplate Fluorescence Reader Fluostar optima.