Characterization of RAW 264.7 Macrophage Cell Line

The RAW 264.7 cell lines (third passage) were purchased from American Type Culture Collection (ATCC® TIB-71™) and from Sigma-Aldrich (cat. no. 91062702). Cells were cultured in DMEM high glucose medium supplemented with 10% FBS (all the experiments were performed using the same FBS batch) and 1% penicillin/streptomycin, in atmosphere of 5% CO₂ and 95% humidity at 37°C. Cells were passaged after reaching 90% confluence, detached with cell scraper and subcultivated in 1:6 ratio in T-75 flasks. All cell culture equipment (flasks, pipettes etc.) used in this study were from the same batch. Cells were cultured continuously from the third passage until passage no. 50. Cells were frozen every fifth passage starting from the passage number three. Before phenotype and functional analysis cells were thawed and cultured for next two passages (e.g. cells of passage number three were thawed and cultured until passage no. 5 and then subjected to further analyses). Cells were regularly tested for Mycoplasma contamination. To avoid differences between culture techniques, cells were cultured only by one person.