Recombinant gm csf

Bone marrow-derived macrophages and dendritic cells were made using six- to eight-week-old Ptgs2^-/- (COX-2^-/-) or C57BL/6 mice as previously described [15 (link),76 (link)]. Briefly, bone marrow was harvested and macrophages were cultured in the presence of M-CSF from transfected 3T3 cell supernatant for six days with a supplement of M-CSF at day three and frozen down for storage. Dendritic cells were cultured in the presence of 20ng/ml recombinant GM-CSF (BD Biosciences, San Jose, CA) for 7 days with a supplement of 20ng/mL GM-CSF every third day. For infection, BMDMs or BMDCs were plated at 1x10⁶ cells/well in a 12 well dish overnight +/- 100ng/mL PAM3CSK4. The following morning, cells were infected with indicated strains of L. monocytogenes or PBS control at an MOI of 10 unless otherwise indicated. Thirty minutes later, supernatant was removed and replaced with medium containing 50μg/mL gentamycin to remove extracellular bacteria. At the indicated times, cells were harvested for western blot or qRT PCR and supernatant was harvested for eicosanoid analysis as described below.