L glutamine

HeLa and HEK293 cells (ATCC) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Merck KGaA), 2 mM L-glutamine (Thermo Fisher Scientific), 100 U/ml penicillin (Meiji Seika Pharma Co, Ltd), and 100 μg/ml streptomycin (Meiji Seika Pharma Co, Ltd) at 37 °C in 5% CO₂. U87-MG cells (provided by Dr Tsuneo Imanaka (University of Toyama)) were cultured in Eagle’s Minimum Essential medium (Nissui) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO₂. Before drug treatment, the culture medium of HeLa cells was changed to DMEM with 0.5% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, and then cells were incubated at 37 °C in 5% CO₂ for 16 to 24 h.

Spleen was collected and squeezed in RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan). Spleen cells were filtered through stainless steel mesh (size, 100 μm). Then, the erythrocytes were lysed with 0.85% NH₄Cl. After washing 3 times with RPMI 1640 medium, the spleen cells were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; JRH Biosciences, Lenexa, KS), 0.03% L-glutamine (Wako Pure Chemical Industries, Osaka, Japan), 1×Antibiotic-Antimycotic (Gibco; Thermo Fisher, Waltham, MA), and were maintained in 5% CO₂ incubator at 37°C. RAW264.7 cells, a mouse macrophage cell line, were cultured at 37°C under 5% CO₂ in Dulbecco’s modified Eagle medium (DMEM; Nissui Pharmaceutical Co.), supplemented with 10% FCS, 0.03% of L-glutamine and 1 × Antibiotic-Antimycotic. For cytokine assays, the spleen cells and RAW264.7 cells were seeded at 2 × 10⁶ cells/well in a 24-well culture plate and incubated at 37°C, 5% CO₂ with 0–5 μg/mL SaEVs for 72 h. The culture supernatants were harvested and stored at −80°C until the cytokine assays were performed.