1α,25(OH)₂D₃ inhibits the oxidative burst in IFN-γ–activated macrophages. (A) Intracellular production of superoxide anion was measured by the conversion of NBT into formazan. BMDMs grown on coverslips were cultured in the presence or absence of IFN-γ and 1α,25(OH)₂D₃. NBT (0.1%) with or without PMA was added for 1 hour and cells were subsequently fixed. Shown is a representative of 3 independent experiments. Original magnification × 100. (B) Single cells from panel A demonstrate specific intracellular formazan precipitation after cellular activation with PMA or IFN-γ. Original magnification × 200. (C) Quantification of O₂^{- ·} production. BMDMs were cultured and NBT/PMA was added as described in panel A. The OD₆₅₀ of cell homogenates was determined photometrically. (D) BMDMs were cultured with IFN-γ and different concentrations of 1α,25(OH)₂D₃. NBT was added and O₂^{- ·} production was quantified as described. Data are presented as the mean ± SEM calculated from triplicate wells; experiments were repeated twice with similar results (C-D). (E) Semiquantitative Cybb RT-PCR. BMDMs were incubated with IFN-γ and VitD₃ as described. Cybb- and β-actin–specific primers were used for amplification. Shown is a representative of 3 independent experiments. (F) Real-time Cybb RT-PCR. RNA for analysis was prepared from BMDMs described in panel E and mRNA expression was normalized as described (= relative mRNA expression).