Bone marrow is harvested from femurs and tibias of C57BL/6 mice. Bone-marrow-derived dendritic cells (BMDCs) are differentiated DMEM. Cultures are differentiated for 7 d and routinely analyzed for >90% CD11c (allophycocyanin (APC) anti-CD11c clone HL3) positivity by flow cytometry before use in experiments. Lentiviral transduction of bone marrow cultures is conducted by addition of 293T culture supernatants containing lentiviral particles encoding the CREB-dependent luciferase reporter construct or CRTC3 targeting or control shRNAs 1 d postisolation. Stable integration of lentiviral shRNA constructs is selected by addition of puromycin (3 μg/mL) on day 4 posttransduction. After 2 d, stably transduced BMDCs are released from selection and used in subsequent assays. Unless otherwise indicated, cells are treated for 2 d with PGE2 (5 μM) or HG-9-91-01 (0.5 μM) or an equivalent concentration of DMSO (≤0.5%) and then stimulated for 18 h with LPS (100 ng/mL), R848 (10 μg/mL), or Zymosan (4 μg/mL)[2].
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
