Gm csf

Purified monocytes were cultured at a density of 1 × 10⁶ cells per mL in complete medium in the presence of recombinant GM-CSF (5 ng/mL), M-CSF (25 ng/mL), IFN-γ (10 ng/mL), IL-10 (10 ng/mL), and IL-4 (800 U/mL); all from R&D Systems, to generate macrophages. For dendritic cell differentiation, monocytes were cultured in the presence of GM-CSF (800 U/mL) in combination with IL-4 (500 U/mL), or GM-CSF (800 U/mL) in combination with IFN-α2a (1,000 U/mL, Cell Sciences). Monocytes were differentiated for 7 days at 37°C in a 5% CO₂ incubator. At day 3, medium was refreshed with the same concentration of recombinant proteins. Cells were harvested at day 7, after 5 min incubation with accutase (Sigma-Aldrich). Next, LAIR-1 expression was determined using flow cytometry, together with the expression of CD14, CD11c, CD163, CD64, CD1a, and CD80, to assess the markers for macrophage and DC differentiation (Supplementary Figure 1).

Healthy volunteer blood samples were collected from Hong Kong Red Cross Blood Transfusion Service according to a protocol approved by the Institutional Review Board of the University of Hong Kong. Human peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats as we previously described [29 (link)]. MDMs were differentiated from PBMCs by providing RPMI-1640 supplemented with 10% FBS, 100 units/mL of penicillin, 100μg/mL of streptomycin, 1% sodium pyruvate, 1% non-essential amino acids, 1U/mL GM-CSF (Cell Sciences, Canton, MA, USA) [30 (link)]. MoDCs were differentiated and maintained as the same condition as MDMs with IL-4 (R&D systems) [31 (link)].