Raw 264

The immunomodulatory activity of L. reuteri INIA P572 was studied in vitro using the murine macrophage cell line RAW 264.7 (European Collection of Authenticated Cell Cultures; ECACC, Salisbury, UK). RAW 264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with heat inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin (all from Sigma-Aldrich). Cells were routinely cultured at 37 °C in a HF160W incubator (Heal Force, Burwood, Australia) with a humidified 5% CO₂ atmosphere. L. reuteri INIA P572 bacterial suspension was prepared in DMEM with gentamicin (Lonza, Barcelona, Spain) at 10⁵ cfu/mL. Before cell stimulation, RAW 264.7 cells were scraped, cell suspension was adjusted to 10⁶ cells/mL and 200 µL of the suspension was incubated in each well in a 96-well plate, and were incubated for 18 h before the experiments.
Cell viability of RAW 264.7 cells after L. reuteri INIA P572 incubation was assessed by the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sigma-Aldrich) as previously described [42 (link)]. Cell viability (%) was calculated by comparing sample absorbance values with untreated control cultures.

The mouse mononuclear macrophage line RAW264.7 was purchased from the China Typical Culture Collection Center. RAW264.7 cells were cultured in DMEM (Sigma‒Aldrich, St. Louis, Missouri, USA) substrate containing 10% FBS. Then, 100 ng/mL lipopolysaccharide (LPS) (Sigma‒Aldrich, St. Louis, Missouri, USA) for one day to induce the polarization of RAW264.7 cells.

Murine carcinoma cancer cell line 4T1 and murine macrophage cell line RAW264.7 were purchased from American Type Tissue Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific) supplemented with 10% feral bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin/streptomycin (PS, Thermo Fisher Scientific) in a humidified incubator at 37 °C with 5% CO₂. Exosomes-free FBS were prepared by ultracentrifugation at 120,000g at 4 °C for 16 h. Cells were routinely tested and mycoplasma-free.
When RAW264.7 cells (naïve M0 macrophages) reached about 60% confluence, they were stimulated with 100 ng/mL LPS (Sigma-Aldrich) and 20 ng/mL IFN-γ (Sigma-Aldrich) for 24 h to induce M1 macrophage polarization. RAW264.7 cells were treated with 20 ng/mL of IL-4 (Sigma-Aldrich) to induce M2 macrophage polarization.