Labsystems multiskan ms microplate reader

Cell viability was assessed using the MTT assay. Cells were seeded into 96-well plates at a density of 5 × 10³ cells/well for H1975 and PCS201-010 as well as of 3 × 10³ cells/well for A549. After overnight incubation, cells were treated with MGs at 10 and 100 μM for 48 h. Note that, due to the low solubility of MG4 and MG10, the highest prepared concentration was 50 μM. Subsequently, the MTT solution (5 mg/mL) was added and then incubated for 4 h. The medium was removed and 150 µL of DMSO was added to each well. Finally, the absorbance of formazan product was measured at a wavelength of 570 nm using a LabSystems Multiskan MS microplate reader (Thermo Scientific, Vantaa, Finland). The selectivity index (SI) was calculated according to the following equation: SI = IC₅₀ for normal cells/IC₅₀ for cancer cells.

Cell-bound and shed forms of syndecan-1 were quantified using the CD138 ELISA Kit (Diaclone Research, Besancon, France) according to the instructions of the manufacturer. Conditioned, serum-free media were concentrated using Centricon centrifugal filter devices with 10 kDa nominal molecular weight limit filters (Merck Millipore, Burlington, MA, USA). Cells were lysed in a buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.5% v/v Triton X-100, and protease inhibitor cocktail (Sigma-Aldrich). The protein content of the samples was determined by Coomassie reagent (Bio-Rad Laboratories Inc.) and 300 μg total protein from each sample was applied to the ELISA plate. Optical densities were read at 450 nm in a LabSystems Multiskan MS microplate reader (Thermo Fisher Scientific).