Mem with non essential amino acids

Monocytes (500,000 cells/well) were added to 24-well tissue culture (TC) plates (Corning Costar, New York, NY, USA) and incubated for 24 h at 37°C in RPMI 1640 – Glutamax – HEPES medium (Gibco, Bleiswijk, The Netherlands) supplemented with 10% FBS, 1% MEM with non-essential amino acids (Gibco), 1% Na-pyruvate (Gibco), 1% Pen/strep (Gibco) with or without 50 ng/ml macrophage colony-stimulating factor (M-CSF) (R&D systems, Minneapolis, MN, USA) in a total volume of 1 ml for 24 h at 37°C. Next, monocytes were stimulated by adding fresh medium, 5 µg/ml β-glucan, 10 ng/ml LPS (LPS derived from Escherichia coli O111:B4, Sigma Aldrich) or a combination of 5 µg/ml β-glucan and 10 ng/ml LPS with or without 50 ng/ml M-CSF (see Figure 1). After 24 h of stimulation, cells were washed once with pre-warmed medium, after which culture medium with or without 50 ng/ml M-CSF was added for another 5 days to start monocyte differentiation. At day 7, macrophages were stimulated for 24 h with 10 ng/ml LPS, after which the supernatant was collected and stored at -20°C for further analysis (see Figure 1). Alternatively, during the 5 day incubation period, monocytes were exposed to 5 µg/ml β-glucan and 50 ng/ml of M-CSF and the medium was collected and stored at -20°C for further analysis.

Dulbecco’s modified Eagle’s medium (low glucose) with L-glutamine (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), hydrogen peroxide, hydrochloric acid, and 100% w/v of trichloroacetic acid solution (TCA) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Fetal bovine serum (FBS) was obtained from ICN Biomedicals (Costa Mesa, CA, USA). Minimum essential medium (MEM) with non-essential amino acids was obtained from Gibco™ (Co Dublin, Ireland). Penicillin–streptomycin was purchased from Nacalai Tesque (Kyoto, Japan). Trypsin–EDTA solution, L-cystine dihydrochloride (Cys2), and fluorescein isothiocyanate–dextran 4 kDa (FD4) were purchased from Sigma Chemical Company (St. Louis, MO, USA).