Poly ic pic

Mouse and human IL-1α, IL-1β and IFNγ were purchased from PeproTech (Rocky Hill, NJ) and used at 10 ng/ml unless stated otherwise. Lipopolysaccharides (LPS), both the smooth strain (S-form) from Escherichia coli 0111:B4 and the rough strain (R-form) from Escherichia coli EH100 (Ra mutant), as well as and poly IC (PIC) were purchased from Sigma-Aldrich (St. Louis, MO). The smooth form of LPS was used for experiments, unless stated otherwise. For standard cell stimulation, LPS was used at 100 ng/ml and poly IC at 10 μg/ml. All culture treatments were done in DMEM containing 0.5% FCS (low serum medium). Cells/culture supernatants were collected at 6 h and 22 h after cell stimulation for real-time PCR, western blot and ELISA, and at 1 day (D) - 3 D for Griess reaction, unless stated otherwise.

Bone marrow stem cells were cultured as previously described in RPMI 1640 medium with 2.05 mM L-glutamine supplemented with 1 μg/ml amphotericin B, 50 IU/ml penicillin, 50 μg/ml streptomycin, 10% heat-inactivated fetal bovine serum (FBS), and 20 ng/ml granulocyte macrophage-colony stimulating factor (GM-CSF) (Life Technologies, Carlsbad, CA) (57 (link)). For cells cultured beyond 8 days, GM-CSF was reduced to 10 ng/ml. Where indicated, cells were stimulated on day 8 of culture with addition of toll-like receptor (TLR) ligands lipopolysaccharide (LPS) (100 ng/ml) (Sigma-Aldrich, St. Louis, MO) or poly I:C (PIC) (50 μg/ml) (Sigma-Aldrich). Cells were harvested on days 10 to 12 of culture by fraction — non-adherent and lightly adherent cells comprised the dendritic cell fraction, while firmly adhered cells dislodged with a cell scraper comprised the Mφ fraction. Mixed bone marrow cultured cell (BMCC) populations included both adherent and non-adherent fractions. Previous research has demonstrated the fractions are distinct populations, although phenotypes overlap somewhat (57 (link)–59 (link)). Our own experiments consistently showed nearly 100% CD11b positivity and slightly less CD11c positivity in all bone marrow populations (some data not shown). A previous study indicates that few neutrophils should be present (57 (link)), consistent with our observation based on morphology.