Bone marrow was harvested from mouse femurs by flushing through with complete RPMI medium and the cells were treated with 1 ml of 0·83% NH4Cl for 3 min to lyse the red blood cells. The cell suspension was plated out at 5 × 105 cells/ml (1 ml/well) in the wells of 24-well plates, in RPMI-1640 medium containing 20% (v/v) GM-CSF. Cultures were stimulated with Poly I, Poly I:C, LPS, CpG ODN, Jap, X31 or PR8, as indicated above, and incubated at 37° for 6 days. Experiments over a time course from 6 to 9 days were initially
undertaken, and a culture period of 6 days was selected because the cultures demonstrated an effect that was not increased over longer time-periods of culture. Surface antigen staining was performed using either directly conjugated mAb or biotinylated mAb followed by staining with phycoerythrin (PE) or Cy-chrome-conjugated streptavidin
(both from BD Biosciences Pharmingen, Oxford, UK). R428 manufacturer The following antibody conjugates were used: mouse CD11c–PE or CD11c–biotin, Gr1–fluorescein isothiocyanate Selumetinib price (FITC) or Gr1–PE (Caltag, Buckingham, UK), B220–allophycocyanin (APC), CD19–biotin (BD Biosciences Pharmingen) and PDCA–biotin (Miltenyi Bergisch Gladbach, Germany), and a mAb to major histocompatibility complex class II (MHCII) (KB6, a gift from Dr M. Parkhouse, Department of Infection and Immunity, Instituto Gulbenkian de Ciencia) was purified and coupled to FITC using standard methods. Fluorescence was analysed using a FACSCalibur flow cytometer (Becton Dickinson, Oxford, UK). Cells were stained with haematoxylin and eosin, and morphological analysis was performed under a light microscope at 400× magnification. Cells were counted in five fields of view and the numbers of different cell types were assessed. Neutrophil-like cells were defined as cells with cytoplasm that stained neutral pink and a multilobed nucleus. Cells containing a large oval nucleus surrounded by a voluminous
cytoplasm were classed as monocytes, and cells containing a large, dark nucleus, with little or no cytoplasm, were classed as lymphoid. Photographs were taken at 200× P-type ATPase magnification using a Canon Powershot G3 mounted onto a Nikon TMS-F inverted microscope. To examine the effects of TLR ligands (representing bacterial and viral PAMPs) and inactivated influenza viruses on the generation of BMDCs, BALB/c bone marrow was cultured in the presence of GM-CSF, with or without the inactivated influenza A viruses Jap (H2N2), X31 (H3N1), or PR8 (H1N1), the TLR3 ligands Poly I and Poly I:C, the TLR4 ligand LPS or the TLR9 ligand CpG ODN. The production of BMDCs was determined by assessing the surface expression of CD11c and MHCII by flow cytometry. The results (Fig. 1a) showed that the addition of influenza virus to BMDC-generating cultures resulted in a marked reduction in the proportion of cells expressing the expected CD11c+ /MHCII+ phenotype. The addition of ligands for TLR3, TLR4 and TLR9 (Fig.