Anti αv 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).

The MCECs were seeded in 48-well or 96-well plates coated with or without PCM-DM. After 24 h, the mean cell areas were evaluated by Image J software (U.S. National Institutes of Health, Bethesda, MD) following actin staining. The numbers of adhered cells after 1 h of seeding were measured with a CellTiter-Glo^TM luminescent cell viability assay (Promega Corporation, Madison, WI) according to the manufacturer's instructions. The number of adhered MCECs at 1 h after seeding was then measured using a Veritas^TM microplate luminometer (Promega Corporation). To elucidate the effect of the interaction between the integrins of the CECs and the PCM-DM on cell adhesion, the MCECs (1×10⁴ cells/well) were seeded in 96-well plates coated with PCM-DM in the presence or absence of integrin-neutralizing antibodies (2 µg/mL), anti-α₁ integrin (Merck Millipore, Billerica, MA), anti-α_v integrin (Merck Millipore), and anti-β₁ integrin (R&D systems Inc., Minneapolis, MN). After 3 h of seeding, adherent cells were measured with the CellTiter-Glo^TM luminescent cell viability assay.