Cyquant nf dye

ASM cells were grown to approximately 50% confluence in 96-well culture plates, and were exposed to serum- and antibiotic-free medium for 24 h. Proliferation of ASM cells over 48h was assayed using the CyQuant NF dye (Invitrogen) as described previously [20 (link)]. Dye calibrations were performed empirically using different cell counts and used to obtain estimates of baseline proliferation and with drug exposures over the 48 h period. Extent of proliferation was normalized to baseline (i.e. time zero).

Ninety six-well non- tissue culture treated plates (Falcon Becton Dickinson, Franklin Lakes, NJ) were coated with fibronectin (Sigma-Aldrich), recombinant CTGF (ProSpec, Ness-Ziona, Israel), or 1% BSA (Fisher Scientific, Pittsburgh, PA) in PBS and were left to dry in a tissue culture hood. To block non-specific binding sites in coated wells, 1% BSA was added to the wells and the plates were incubated at 4°C for 1 hour. BSA was discarded and wells were washed with 1X PBS prior to adding 2 x10⁴ MC3T3-E1 cells (passage 5 to 10) to the wells and incubation at 37°C for 45 minutes. To block the various integrins of interest, cells were incubated with 5 μg/ml BioLegend (San Diego, CA) monoclonal integrin antibodies against α_v, α₂, α₅, β₁, β₃, β₅ or normal IgG for 30 minutes at 37°C prior to seeding in 96-well plates. Wells were washed with 1X PBS. CyQuant NF dye (Invitrogen, Carlsbad, Ca) was added to each well and the plates were incubated at 37°C for 1 hour. Fluorescence was measured using a microplate reader with excitation at ~485 nm and emission detection at ~530nm. Relative fluorescence units (RFUs) were converted to cell number using standard curve made by performing adhesion assay for different cell numbers.