Hemocytometer counting chamber

The antiproliferative effect of MGR on cancer and normal cells was quantified by MTT assay.47 Briefly, upon reaching 90% confluency, the cell concentration was determined using a hemocytometer counting chamber (Marienfeld, Lauda-Königshofen, Germany). Then, the cells, at 1 × 10⁵ cells/mL, were cultured in 96-well microculture plates (TPP, Trasadingen, Switzerland) and treated with various concentrations of MGR in triplicates. MTT solution (Sigma-Aldrich) was added to the cells after incubation for 72 hours at 37°C. After exactly 4 hours of incubation in the dark, MTT solubilization solution (Sigma-Aldrich) was added and the plate was allowed to stand for 5 minutes at room temperature. The OD was then measured at 570 nm using an ELISA plate reader (Biotech, Inc, Alpharetta, GA, USA). The IC₅₀ value was determined from absorbance versus concentration curve and compared to that of doxorubicin, the commonly used antineoplastic agent (Sigma-Aldrich), and the negative control, 0.1% DMSO (Sigma-Aldrich).

The cell culture and growth inhibition assay were performed as previously reported.14 Briefly, the MCF-7 cells were acquired from the American Type Culture Collection (Manassas, VA, USA) and grown in Roswell Park Memorial Institute medium-1640 supplemented with 10% FBS and 1% penicillin–streptomycin. The cell count for each of the assay was determined using the hemocytometer counting chamber (Marienfeld, Germany), and 0.5–1×10⁶ cells were subcultured into a fresh 25 cm² tissue culture flask containing fresh medium (6 mL) at the subcultivation ratio of 1:4 while the cultures were incubated at 37°C under 5% CO₂ and 95% air.