Anti α5 integrin

The involvement of integrins in the enhancement of cell adhesion by the ROCK inhibitor was evaluated by seeding MCECs (5 × 10³ cells/well) in 96-well plates in the presence or absence of integrin-neutralizing antibodies (2 μg/mL): anti-α1 integrin (Merck Millipore, Billerica, MA), anti-α2 integrin (Merck Millipore, Billerica, MA), anti-α3 integrin (Merck Millipore, Billerica, MA), anti-α4 integrin (Merck Millipore, Billerica, MA), anti-α5 integrin (Merck Millipore, Billerica, MA), anti-α6 integrin (Merck Millipore, Billerica, MA), anti-αV integrin (Merck Millipore, Billerica, MA), anti-α6 integrin (Merck Millipore), and anti-β1 integrin (R&D systems Inc., Minneapolis, MN). The effect of inhibiting phosphorylation of MLC and RhoA activity on cell adhesion was also evaluated by seeding MCECs with blebbistatin (10 μM) and C3 (300 ng/ml), respectively. Three hours after seeding, the numbers of adherent cells were determined with the CellTiter-Glo^TM luminescent cell viability assay (Promega Corporation, Madison, WI) according to the manufacturer’s instructions. The number of adhered cells was determined using a Veritas^TM microplate luminometer (Promega Corporation).