Lps free fbs

Bone marrow was harvested and cultured as previously described[23 (link)]. On day six of the culture the cells were harvested from the plates and semi-adherent cells removed. The cells were spun at 400g and re-suspended at a concentration of 1×10⁶ cells/ml in DC medium (RPMI 1640 supplemented with 10% LPS-free FBS (Gibco, Paisley, UK) 1% Penicillin/streptomycin and 50mM Beta-mercaptoethanol (Sigma Aldrich, Dorset UK)). The cells were stimulated overnight with LPS (100ng/ml) or T. muris antigen (5μg/ml). After 24 hours the cells were harvested and prepared for flow cytometry. FcR were blocked by incubating cells in anti-CD16/32 (2μg/ml) for 15 minutes. Cells were washed and stained with anti-CD45 PeCy7 (1 μg/ml, Becton Dickinson), anti-CD11c Alexa700 (1μg/ml), anti-MHC-II FITC (2.5 μg/ml) and anti-CD86 PE (1 μg/ml) (all E Bioscience) and acquired using a LSRII flow cytometer (Becton Dickinson Biosciences, Oxfordshire). Data was analysed using Flow Jo flow cytometry analysis software (Tree Star, Inc, Oregon, US).