Insulin transferrin selenium solution its

Mouse mesothelioma cell lines were previously generated in our laboratory and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12 + Glutamax; Gibco), supplemented with 4 μg/mL Hydrocortisone (Sigma), 5 ng/ml murine EFG (Sigma), insulin-transferrin-selenium solution (ITS; Gibco), 10% fetal calf serum (FCS; Capricorn), and 1% penicillin and streptomycin (Gibco).¹⁵ (link)^,52 (link) Human mesothelioma cell lines were obtained from the American Type Culture Collection (ATCC) and were cultured in mammalian cell culture medium as specified above. Uveal melanoma cell lines, also obtained from ATCC, were cultured in either RPMI 1640 (RPMI-1640; Gibco) or Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% or 20% FCS and 1% penicillin/streptomycin. All cell lines were maintained at 37°C in a humidified atmosphere containing 5% carbon dioxide (CO₂) and were tested for mycoplasma contamination using MycoAlert Mycoplasma detection kit (Lonza). The human cell lines were authenticated using short tandem repeat STR DNA profiling.

FLCs differentiation from hESC was described above. FLCs on day 11 to day 20 were picked under a dissecting microscope and each FLC was transferred into 20 μl in vitro culture medium (IVCM) using 20 μl pipette tips. IVCM was composed of 5% FBS (Gibco), 0.026 M NaHCO₃ (Sigma), 1 × penicillin and streptomycin solution (Corning), 1 × insulin/transferrin/selenium solution (ITS; Gibco) and 0.01 μg ml⁻¹ follicle-stimulating hormone (FSH; R&D) in MEM-α (Corning) as described previously in an in vitro maturation study of mice follicles36 (link). in brief, each FLC grown on the plate was separated with minimal disturbance to the intact structure, washed twice in IVCM and transferred into 20 μl IVCM attached to the lid of a 60 mm culture dish (Corning). The lid was put in a dish filled with 3 ml of PBS (Corning) to avoid IVCM evaporation. FLC drop culture was incubated at 37 °C in 5% CO₂. The medium was collected and changed every 24 h.