Ham s f12 nutrient medium

CHO-K1 cells (ATCC CCL-61) and CHO-derived cell lines were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 nutrient medium (Sigma N6658) supplemented with 5% fetal bovine serum (Sigma F2442), 2 mM L-glutamine, and 1 mM sodium pyruvate. NIH 3T3 cells (ATCC CRL-1658) were maintained in DMEM with 10% fetal calf serum and 2 mM glutamine. For lipotoxicity experiments, growth medium was supplemented with 500 μM palmitate (Nu-Chek Prep) complexed to BSA (Sigma A8806) at a 2:1 molar ratio, as described previously^{14 (link)}. Normal human skin fibroblasts (NSF, ATCC CRL-1474) were grown in DMEM supplemented with 10% inactivated fetal bovine serum.

Cell viability screening of alkaloid fractions of the ten species of Amaryllidaceae was performed in gastric cancer cell line AGS (CRL-1739^™), prostate cancer cell line PC3 (CRL-1435^™), breast cancer cell lines BT-549 (HTB-122^™), MCF7 (HTB-22^™) and MDA-MB 231 (HTB-26^™), uterine cancer cell line HEC-1B (HTB-113^™) and human keratinocytes HaCat (PCS-200-011^™) as a non-carcinogenic control cell line (all cells were obtained from ATCC^®) [58 (link),59 (link)]. The cells were cultured in Dulbecco Modified Eagle Medium-high glucose (DMEM, Sigma–Aldrich, St. Louis, MO, USA) and Ham’s F-12 nutrient medium (F12, Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated FBS (Sigma–Aldrich, St. Louis, MO, USA), 100 U/mL penicillin (Sigma–Aldrich, St. Louis, MO, USA) and 100 μg/mL streptomycin (Sigma–Aldrich, St. Louis, MO, USA), 2 mM L-glutamine (Sigma–Aldrich, St. Louis, MO, USA) in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. Cells were monitored on a Nikon Eclipse TS100 (Melville, NY, USA) inverted phase contrast microscope. Cell viability experiments were performed when cells reached 75–80% confluence using 0.25% trypsin 1 mM EDTA (Sigma–Aldrich, St. Louis, MO, USA).