Mccoy s 5a modified medium

The osteosarcoma cell lines (MG-63 and Saos-2) were purchased from ATCC (Manassas, VA, USA) and cultivated according to the manufacturer’s recommendation in Dulbecco’s Modified Eagle Medium (DMEM, Corning) and McCoy’s 5A (modified) Medium (Biological Industries) containing 10% fetal bovine serum (FBS), respectively. The media were supplemented with 1% penicillin/streptomycin. The cells were cultured on standard polystyrene plates (NEST Biotechnology) under controlled atmospheric conditions (37 °C temperature, 5% CO₂). The culture medium was changed every 2–3 days. T the cells were sub-cultured after 3–4 days when the confluence reached about 80%. For passaging, the cells were detached with trypsin/EDTA and subsequently seeded. The same density of seeded cells was used in all the experimental approaches. The cells were seeded at 25 × 10³ cells/mL (25,000 cells per disk in a 24-well plate) in a serum-free medium and incubated at 37 °C for an appropriate period of time. Prior to use, the substrates were sterilized by triple soaking in pure ethanol and then triple rinsing in sterile PBS (Corning Life Science and ALAB, Warsaw, Poland). Every 2 days the medium was replaced with a fresh one.

HEK293T cells were cultured in DMEM medium (Thermo Fisher Scientific). Jurkat-NFAT-FcγRIIIa reporter cell line, CD40 reporter cell line and Ramos cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific). SK-BR-3 cells were cultured in McCoy's 5A (Modified) medium (Biological Industries). All the culture medium was supplemented with 10% fetal bovine serum (Biological Industries), 1× non-essential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin and 12.5 mM HEPES (Thermo Fisher Scientific). HEK293F cells were suspension cultured in FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific). CHO-K1 or FUT8^-/- CHO-K1 cells were cultured in FreeStyle™ CHO Expression Medium (Thermo Fisher Scientific). All the cells were maintained in CO₂ incubator at 37 °C.