Rpmi 1640 glutamax hepes medium

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.

The gastric carcinoma cell lines MKN45 (Lauren diffuse-type) and AGS (Lauren intestinal-type) [24 (link)] were obtained from the Japanese Collection of Research Bioresources and ATCC, respectively. The MKN45 and AGS SimpleCell models (MKN45 SC and AGS SC) were obtained by targeting the C1GALT1 (COSMC) gene using zinc-finger nuclease precise gene editing as previously described [21 (link),25 (link)]. Furthermore, MKN45 cell line was stably transfected with the full-length human ST6GALNAC1 gene (MKN45 ST6) or the corresponding empty vector pcDNA3.1 (MKN45 MOCK) as previously described [26 (link),27 (link)]. The MKN45 WT/SC and AGS WT/SC cell lines were cultured in RPMI 1640 GlutaMAX™, HEPES medium (Gibco, Waltham, MA, USA). The MKN45 MOCK/ST6 cell lines were cultured in RPMI supplemented with 0.5 mg/mL of G418 (Invitrogen). All media were supplemented with 10% heat-inactivated FBS (Biowest, Riverside, MO, USA). All cells were grown at 37 °C in an atmosphere of 5% CO₂.

Monocytes (500,000 cells/well) were added to 24-well tissue culture-treated 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 (HyClone), 1% MEM non-essential amino acids (Gibco), 1% Na-pyruvate (Gibco), 1% Pen/strep (Gibco) and 50 ng/ml macrophage colony-stimulating factor (M-CSF) (R&D systems, Minneapolis, MN, USA) as described before (Moerings et al., 2021 (link)). Briefly, resilience was induced by treatment of monocytes with 10 ng/ml LPS (Sigma-Aldrich, Escherichia coli serotype O111:B4) for 24 h, followed by washout and five days exposure to 5 μg/ml arabinoxylan preparation in culture medium with 50 ng/ml M-CSF. Monocytes used to apply the training model were kept in culture medium for 24 h, followed by washout and five days exposure to 5 μg/ml arabinoxylan preparation in culture medium with 50 ng/ml M-CSF. Establishment of resilience or training in the resulting macrophages was determined by stimulation with 10 ng/ml LPS on day 7. After 24 h, supernatants were collected and stored at −20 °C for further analysis.