Cell titer 96 aqueous one solution cell proliferation assay solution

Cellular growth curves were plotted using cellular viability values measured by the MTS method (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay solution; Promega). GC cells transfected with gal-siRNA, NC , galectin-3-overexpression were seeded at 1000/cells per well in 96-well flat-bottomed microtiter plates in a final volume of 200 µL of culture medium per well. Cells were incubated in a humidified atmosphere (37°C and 5% CO2) for 24, 48, 72, 96, 120, 144, 168 hours after transfection. MTS assays were used to measure cell proliferation, with 5 replicates at each time point. Statistical analyses were carried out using the two-tailed unpaired Student's t-test.

Cells were seeded into 96-well plates in 100 µL of media 24 hours after transfection with 6 replicates of each condition. After a further 24 hours, 20 µL of CellTiter 96 AQueous One Solution Cell Proliferation Assay solution (Promega, UK) was added to each well. Colorimetric absorbance was measured at 490 nM on the Wallac 1420 Victor 3 Multilabel Counter plate reader (Perkin-Elmer). Background absorbance was subtracted from media-only wells.

SU-8 2150 epoxy photoresist, SU-8 developer, and Omnicoat were obtained from MicroChem, SUEX dry film epoxy photoresist from DJ Microlaminates, PDMS Sylgard silicone elastomer from Dow Corning, and Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA), penicillin/streptomycin, 4′,6-diamidino-2-phenylindole (DAPI), Live/Dead^® Viability/Cytotoxicity Kit, Alexa Fluor™ 594 Phalloidin, CellTracker™ Green CMFDA Dye, and CellTracker™ CM-DiI from ThermoFisher Scientific. The CellTiter 96^® AQueous One Solution Cell Proliferation Assay solution was purchased from Promega.

Cell viability was determined using the CellTiter 96® AQueous One Solution Cell Proliferation Assay solution (MTS; Promega, WI, USA). The assay is based on the reduction of MTS tetrazolium to a colored formazan dye by metabolically active cells. RWPE-1 and WPMY-1 cells were seeded into a 96-well plate and treated with 3.13 to 200 μM concentration of BC for 24 h. After treatment, BC-treated cells were added along with MTS solution for 4 h, followed by measurement of cell viability by recording the absorbance of formazan dye at 490 nm using a microplate reader (Biotek; VT, USA).

Gelatin from porcine skin (type-A, 300 bloom), methacrylic anhydride, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), Opti-MEM I reduced serum medium (Opti-MEM), fetal bovine serum (FBS), goat serum, trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA), antibiotic-antimycotic solution stabilized (Anti-Anti, 100X), 4′,6-diamidino-2-phenylindole (DAPI), formalin (10% w/v), Live/Dead^® Viability/Cytotoxicity Kit, Alexa Fluor^® 594-phalloidin, dialysis membrane (M_w cutoff: 12–14 kDa) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The CellTiter 96^® AQueous One Solution Cell Proliferation Assay solution was obtained from Promega (Madison, WI, USA). Mouse cytochrome P450 3A (CYP3A4/CYP3A5 monoclonal) antibody and Alexa Fluor^® 594-conjugated goat anti-rabbit secondary antibody were purchased from Abcam (Cambridge, MA, USA). Ruthenium visible light photocrosslinking kit was purchased from Advanced BioMatrix (San Diego, CA, USA). Glass coverslips of 8 mm in diameter were obtained from Thomas Scientific (Swedesboro, NJ, USA). All other chemicals used in this study were obtained from Sigma-Aldrich unless otherwise mentioned.