Mts assay solution

Roswell Park Memorial Institute (RPMI)-1640 (Invitrogen, Carlsbad, CA, USA), fetal bovine serum (FBS) (Invitrogen), 1% antibiotic-antimycotic (AA) solution (Invitrogen), Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Invitrogen), 1,4-dithiothreitol (DTT) (Sigma-Aldrich, St. Louise, MO, USA), radioimmunoprecipitation assay (RIPA) buffer (89900; Pierce), bicinchoninic acid assay (23225; ThermoFisher, Waltham, MA, USA), sample buffer (125 mM Tris pH 6.8, 4% sodium dodecyl sulfate (SDS), 10% glycerol, 0.006% bromophenol blue, and 1.8% mercaptoethanol), precast protein gel (4561094; Bio-Rad, Hercules, CA, USA), primary antibodies, anti-c-Met (ab51067; Abcam, Cambridge, UK; 4560; Cell Signaling, Massachusetts, USA), anti-nucleolin (ab22578/ ab13541; Abcam), anti-HER2 (SC-08; Santa Cruz, TX, USA), anti-GAPDH (SC-47724; Santa Cruz), MTS assay solution (G3585; Promega, WI, USA), μ-Slide 8-well (ibid; München, Germany), Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs), and Matrigel (BD Biosciences, NJ, USA) were used.

RAW 264.7 cell lines were purchased from American Type Culture Collection, Middlesex, UK (ATCC1 TIB-71™). Cells were cultured in DMEM high glucose medium, supplemented with 10% FBS, 2 mM L-Glutamine and 1% penicillin/streptomycin, at 37 °C in a humidified incubator containing 5% CO₂. Cells were passaged after reaching 90% confluence.
Cells, in logarithmic growth, were plated in 96-well plates at a density of 1 × 10⁴ cells/well in 200 µL of media and incubated for 24 h. The media on the cells was removed and replaced with 180 µL of fresh media. To each respective well, 20 µL of test compound at a concentration of 1 mM was added, resulting in a final compound concentration of 100 µM. To control wells, DMSO drug solvent was added. In all wells, the final concentration of DMSO did not exceed 0.1%. Following incubation for a further 96 h at 37 °C in a humidified incubator containing 5% CO₂, all media was removed and replaced with 100 µL of fresh media. To each well, 20 µL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay solution (Promega, Madison, WI, USA) was added, and the plate was incubated in the dark at 37 °C for 4 h. Finally, the absorbance of each well was recorded at 490 nm using a microplate reader. The percentage of cell viability was calculated relevant to the compound solvent (DMSO) control.