Isolation and characterisation of macrophages from 3 different compartments. (A) Mononuclear cells were harvested from the lungs, blood and bone-marrow. A large number of cells (alveolar macrophages, PBMC and bone-marrow cells) were harvested from the 5 breeds of pigs (Duroc, Piétrain, Landrace, Hampshire and Large White). (B) The 3 type of macrophages were stimulated with LPS in order to analyse the inflammatory response. Monocyte and bone-marrow cells were cultured 5–6 days with rhCSF-1 (1×106 units/ml) in order to obtain macrophages. AM (Δ), BMDM (□) and MDM (○) were plated at 1×106 cells/ml and left untreated (black) stimulated with 100 ng/ml of LPS (white). Supernatant was harvested at different timepoints (0, 2, 6, 10, 24, 30, 48 and 54 h) and porcine TNF was measured by ELISA (pg/ml). Morphology of AM, MDM and BMDM are pictured in (C, D and E, respectively) after being left overnight in complete medium with rhCSF-1. Surface expression of CD14 (F-G-H) and CD16 (I-J-K) on AM, MDM and BMDM was measured by flow cytometry. 1×106 cells were stained and data was acquired from 15,000 events. The correct isotype control is shown by a dark black line and the targeting antibody by the lighter line. Data is representative of a minimum of 3 different experiments. (L) TNF production (pg/ml) of AM, MDM and BMDM at 0 and 7 h of LPS stimulation in function of the 5 breeds of pigs (DUR, PIE, LAN, HAM and LW). Histograms are the average of 5 individual pigs +/- SED. For AM, TNF production from DUR (a) and PIE (b) are significantly lower than HAM and LW (p<0.05).