Hff 1

Human epithelial colorectal adenocarcinoma cells (Caco-2, HTB-37) and human foreskin fibroblasts (HFF-1, SCRC-1041) were obtained from LGC Standards (Teddington, Middlesex, UK). Caco-2 cells were cultured according to manufacturer’s instruction in DMEM hg w/L-glu medium supplemented with 10% v/v FBS and penicillin-streptomycin solution at 37 °C in a humidified atmosphere of 5% CO₂. HFF-1 cells were cultured in DMEM hg w/L-glu/sp medium supplemented with 15% v/v FBS and penicillin-streptomycin solution at 37 °C in a humidified atmosphere of 5% CO₂.

Human fibroblasts (HFF-1, ATCC-SCRC-1041, LGC Standards, Poland) were grown in DMEM (Biowest, Riverside, MO, USA) containing high glucose, supplemented with 15% (v/v) heat-inactivated FBS (Biological Industries, Bet ha-Emek, Israel) and 1% (v/v) penicillin/streptomycin mixture (Biowest, USA), at 37 °C in a humidified atmosphere of air with 5% CO₂ for 3 days. A confluent monolayer of the cells was detached with trypsin (Biowest, USA) and cell suspensions at 1 × 10⁶ cells·mL⁻¹ were seeded into 96-well tissue culture plates (Nunc, Roskilde, Denmark) at 100 µL/well for 48 h incubation at 37 °C as above. The culture medium was replaced with 100 µL medium containing the fractionated SBT extracts over a concentration range of 0.007–1.0 mg·mL⁻¹ for 24 h incubation, with appropriate positive and negative controls being set up at the same time. The pro-proliferative/cytotoxic activity of the fractions was measured by MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay [48 (link)]. Final absorbance of the samples was read at 550 nm with a Victor² microplate reader (Wallac, Turku, Finland). The results for the test samples and the controls were used to calculate % cell viability and the IC₅₀ (concentration giving 50% loss of viability).