Glutamin

Osteoblast-like cells were isolated from long bones of 6 weeks old male mice (n = 4). After removal of the muscle and soft tissue, the bones were washed with sterile PBS and cut longitudinally into 1 mm long fragments. The fragments were incubated in 6-well plates (Falcon) in αMEM Medium (Lonza, Verviers, Belgium) supplemented with 10% FBS (Lonza, Verviers, Belgium), 1% penicillin/streptomycin (Biochrom, Berlin, Germany), and 5 mM glutamin (Biochrom, Berlin, Germany) for 21 days (humidified incubator, 37 °C, 5% CO₂) to allow cell outgrowth. Media was exchanged weekly. After reaching confluency, the cells were trypsinized and sub-cultured in 1:4 ratio. For immunostaining, cells were plated onto coverslips in 24 well plates. The commercial rat osteosarcoma cell line (UMR 106, American Type Culture Collection ATCC, Manassas, VA, RRID:CVCL 3617) was used as control.

Splenic B cells from MD4 mice were enriched using MACS technique as described above. B cells were cultured for 24 hours in RPMI medium (Biochrom, Berlin, Germany) with 10% FCS (GE Healthcare Life Sciences, Buckinghamshire, UK), 1% Pen/Strep (Gibco, Thermo Fisher Scientific, Waltham, USA) and 0,3mg/ml Glutamin (Biochrom) containing 1μg HEL (Sigma) for stimulation. Subsequently, cells were i.v. injected into WT mice, which were simultaneously orally gavaged with 1mg HEL or PBS. One day later mice were sacrificed and analysed via flow cytometry.

Cell suspensions from the spleen of B6-Il10^-/- mice were made and erythrocytes were lysed as described previously (23 (link)). Splenic T cells were isolated by negative selection using magnetic cell separation. Therefore, cell suspension was incubated with anti-MHCII (BD Biosciences) and IgG beads (MACS, Miltenyi) each for 20 min. Isolated cells were stained with CFSE (5 mmol) for 2 min and washed with MACS buffer. Next, 2x10⁶ cells/well were cultured with or without anti-CD3 antibody (Biolegend) for 48 hours in RPMI medium (Biochrom, Berlin, Germany) with 10% FCS (GE Healthcare Life Sciences, Buckinghamshire, UK), 1% Pen/Strep (Gibco, Thermo Fisher Scientific, Waltham, USA), 0.3 mg/ml Glutamin (Biochrom), and 10 µM β-mercaptoethanol containing recombinant protein (770 ng/mL CCL5/RANTES 134 ng/mL CCL2, 200 ng/mL CCL7, and 150 ng/mL CXCL16; all Peprotech) for stimulation. Stimulation experiments were performed in duplex and data were generated from 2–5 independent experiments.