Stable glutamine without sodium pyruvate

For infection experiments murine RAW264.7 macrophages (American Type Culture Collection, ATCC no. TIB-71), RAW264.7 macrophages stably transfected with LAMP1-GFP or primary human monocytes were used. RAW264.7 macrophages were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 3.7 g × l⁻¹ NaHCO₃, 4.5 g × l⁻¹ glucose, 4 mM stable glutamine without sodium pyruvate (Biochrom) and supplemented with 6% iFCS (Sigma-Aldrich). Primary human monocytes were cultured in RPMI medium containing 5.5 g × l⁻¹ NaCl, 5.0 mg × l⁻¹ phenol red, 2.0 g × l⁻¹ NaHCO₃, 25 mM HEPES, 4 mM stable glutamine without sodium pyruvate (Biochrom) and supplemented with 10% iFCS (Sigma-Aldrich).

Buffy coat was obtained via the blood bank of Deutsches Rotes Kreuz (Springe, Germany) from pooled samples of voluntary, anonymous blood donors who gave informed consent. Lymphocytes were prepared from buffy coat by Ficoll-Hypaque density gradient centrifugation⁵⁷ . Buffy coat was diluted in PBS (ratio 1:3) and Ficoll-Hypaque was added, following centrifugation for 20 min at 800×g. Afterwards, 5 × 10⁸ cells were cultured in Roswell Park Memorial Institute (RPMI) medium containing 5.5 g × l⁻¹ NaCl, 5.0 mg × l⁻¹ phenol red, 2.0 g × l⁻¹ NaHCO₃, 25 mM HEPES, 4 mM stable glutamine without sodium pyruvate (Biochrom), 100 units × ml⁻¹ penicillin, 100 µg × ml⁻¹ streptomycin, 2.5 ng × ml⁻¹ GM-CSF (granulocyte-macrophage colony-stimulating factor) and supplemented with 10% human plasma. After o/n incubation at 37 °C in an atmosphere of 5% CO₂ and 90% humidity, non-adherent cells were removed, and adherent cells were cryo-preserved in inactivated fetal calf serum (iFCS) and DMSO. Next, cell suspensions were thawed, seeded, and maintained in RPMI-1640 (Biochrom), supplemented with 20% FCS and 2.5 ng × ml⁻¹ GM-CSF (Peprotech) to differentiate monocytes into macrophages. After 5–7 days, the purity of the macrophage population was checked by staining with FITC anti-human CD14 antibody (BioLegend) and FC analyses. The macrophages were used for infection by STM strains.