Calcitriol

Viable monocytes were cultured at a density of 1 × 10⁶ cells/mL in 24-well plates in IMDM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 2% heat-inactivated human AB serum (Sigma-Aldrich, St. Louis, MO, USA), 250 U/mL IL-4 and 200 U/mL granulocyte macrophage colony-stimulating factor (GM-CSF) (both from Peprotech, London, UK) for 6 days at 37 °C and 5% CO₂ atmosphere. On day 4, culture cell medium and cytokines were refreshed and a maturation cocktail containing 1000 U/mL IL-1β, 1000 U/mL TNF-α (both from Peprotech) and 1 μM prostaglandin-E2 (PGE2) (Pfizer, New York, NY, USA) was added to obtain mDC. For the generation of VitD3-tolDC, monocytes were additionally treated with 1 nM 1α,25-dihydroxyvitamin D3 (Calcitriol, Kern Pharma, Barcelona, Spain) on days 0 and 4. On day 6, cells were harvested by 25 min of accutase incubation at 37 °C to detach the cells from the plate. After washing, cell counts and viability were assessed by flow cytometry as previously mentioned, and phenotype and functional characterization were determined (see below). Dry pellets of each condition with the remaining cells were stored at −80 °C.

Rat osteosarcoma cells UMR 106 (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Biowest, Riverside, MO, USA). Cells were seeded in 6 wells plates with 10,000 cells/cm² and maintained in DMEM up to 90% confluence. FGF23 production was stimulated by adding calcitriol (10⁻⁸ M) (Kern pharma, Barcelona, Spain), as previously reported [25 (link)]. Two groups of experiments were performed to study the effect of acidosis on FGF23 production: short-term and longer-term experiments. Short-term experiments were carried out in cells that had grown in a medium with normal (7.4) pH and were briefly exposed (for 24 h) to a low (7.2) pH medium. The pH of the culture medium was reduced by adding HCl. In longer-term experiments, cells were incubated until they reached confluence (for 6 days) in either DMEM with normal pH (7.4) or DMEM with low pH (7.2).