G 5011

Human macrophages were differentiated at day 5, and activated with IL-4 (20 ng/ml) for additional 48 h. At day 7, the M(IL-4) macrophages were washed with warmed RPMI-1640 medium supplemented with M-CSF (10 ng/ml) and pre-incubated for 30 min with 10 µg/ml blocking antibodies specific to either Dectin-1 (MAB1859, R&D Systems) or DC-SIGN (ab13847, abcam), or both. As a control, M(IL-4) macrophages were incubated with an irrevelevant antibody (Ab-Control). For efficient stimulation of Dectin-1, M(IL-4) macrophages were treated with cytochalasin D (1 µg/ml, C8273, SIGMA) in combination with purified β-glucan from Saccharomyces cerevisiae (10 µg/ml, G5011, SIGMA), as previously described (31 (link)); for DC-SIGN, cells were stimulated with 10 µg/ml of ManLAM (kindly provided by Dr. Jerome Nigou, IPBS/CNRS); and for TLR-4, cells were stimulated with 1 µg/ml of LPS. After 24 h, the supernatants were collected and stored at −80°C until further use for ELISA analysis.

β-glucan used in this study was purchased from Sigma (G-5011), which was hereinafter referred to as β-glucan for short. β-glucan with a β-(1,3)-glucan linear structure and a small number of β-(1,6)-glucan branches was extracted from Saccaromyces cerevisiae and had been used in other researchers' studies 31 (link), 32 (link). The preparation method and characterization of β-glucan has been described in the previous studies 22 (link), 33 (link), and the purity of β-glucan is greater than 98%.