Stable glutamine

The murine macrophage line RAW264.7 (American Type Culture Collection; ATCC no. TIB-71) was cultured in high-glucose (4.5 g · ml⁻¹) Dulbecco’s modified Eagle’s medium (DMEM) containing 4 mM stable glutamine (Merck) and supplemented with 6% inactivated fetal calf serum (Sigma). The nonpolarized epithelial cell line HeLa (ATCC no. CCL-2) was cultured in high-glucose DMEM containing 4 mM stable glutamine and sodium pyruvate and supplemented with 10% inactivated fetal calf serum. Stably transfected HeLa cell lines expressing LAMP1-GFP or LifeAct-GFP were cultured under the same conditions. All cells were maintained at 37°C in an atmosphere containing 5% CO₂ and absolute humidity.

Human OSCC cell line PCI-13 was obtained from the University of Pittsburgh Cancer Institute (UPCI, Pittsburgh, PA, USA) [27 (link)]. CD34+, c-kit+, and CD13+ human MC cell line LUVA was obtained from KERAFAST (Kerafast, Boston, MA, USA). PCI-13 cells were cultured in MEM with Earle’s salts, 2.2 g/L NaHCO3, stable glutamine, and low endotoxin (Merck, Darmstadt, Germany), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Biochrom, Berlin, Germany), 1% nonessential amino acids (NEAA) (PAN Biotech, Aidenbach, Germany), 100 U/mL penicillin, and 100 μg/mL streptomycin (PAN Biotech), and incubated in a humidified chamber with 5% CO₂ at 37 °C. Human MC suspension cell line LUVA was cultivated in 20 mL of StemPro™-34 SFM medium (Thermo Fisher Scientific, Waltham, MA, USA) in upright T175 flasks and incubated in a humidified chamber with 5% CO₂ at 37 °C.

The murine macrophage cell line RAW264.7 (American Type Culture Collection, ATCC no. TIB-71) was cultured in high-glucose (4.5 g x ml^-1) Dulbecco’s modified Eagle’s medium (DMEM) containing 4 mM stable glutamine (Merck) and supplemented with 6% inactivated fetal calf serum (iFCS, Sigma). The human macrophage-like cell line U937 (ATCC no. CRL-1593.2) was cultured in RPMI-1640 medium (Merck) supplemented with 10% iFCS. Human primary macrophages were generated from monocytes isolated from buffy coat as previously described [50 (link)], and cultured in RPMI-1640 medium supplemented with 20% iFCS. Under these conditions, predominantly M1-polarized macrophages were obtained. The non-polarized epithelial cell line HeLa (ATCC no. CCL-2) was cultured in high-glucose DMEM containing 4 mM stable glutamine, sodium pyruvate and supplemented with 10% iFCS. Stably transfected HeLa cell lines expressing LAMP1-GFP were cultured under the same conditions. All cells were cultured at 37°C in an atmosphere containing 5% CO₂ and absolute humidity.