BM-DCs generation and treatment

Bone marrow cells were cultured with recombinant murine GM-CSF (125 U/ml, Pepro Tech Inc., Rocky Hill, NJ) and 2-mercaptoethanol (50 μM) for 6 days as described previously [17]. Day-6 BM-DCs were further purified with anti-mouse CD11c magnetic beads (Miltenyi Biotec, Sunnyvale, Calif., USA) according to the manufacturer’s instructions. Purified BM-DCs were treated with different concentrations (0–50 μM) of DMSO, Zinc protoporphyrin-IX (ZnPP), Tin protoporphyrin-IX-chloride (SnPP) or cobalt (III) protoporphyrin-IX-chloride (CoPP) for 2 hours and replaced with fresh medium for further 14 hours. LPS (1 μg/ml, Escherichia coli O127:B8; Sigma-Aldrich, St. Louis, Mo., USA) was then added as stimuli and cultured for 24 hours. The cells were harvested for phenotypic analysis, western blotting or the analysis of T-cell responses, and supernatants collected for cytokine determination by ELISA (eBioscience, Ireland, UK). The phenotype and purity of BM-DCs were analyzed by flow cytometry (LSR II; BD Biosciences, San Diego, Calif., USA) for the expression of CD11c (G418), MHC class II (M5/114.15.2), CD40 (1C10), CD80 (16-10A1) and CD86 (GL1).