Power wave 340 plate reader

Cytokine levels in cell culture supernatants were measured with the help of commercially available ELISA kits detecting IL-2, IL-6 or TNFα, following the instructions of the manufacturer (Biolegend). Samples were pre-diluted, depending on the experimental setup, to account for the different cytokine concentrations in cocultures of T cells and monocytes with the two tumor cell lines. Absorbances at 450 nm and 570 nm were recorded using a BioTek Power wave 340 Plate Reader (BioTek Instruments, Germany, Wetzlar).

One million BON and NCI-295R cells per well were seeded in 6-well plates and treated for 2 and 6 h, respectively, with TNFα on the following day. Cells were lysed in the RIPA buffer containing Complete Mini Protease Inhibitor Cocktail (Roche, Basel, Switzerland). The protein concentration was measured using the BCA kit (Thermo Fisher) with the PowerWave340 plate reader (Biotek, Winooski, VT, USA). With 10 µg of proteins loaded per well on the 3.9–20% gel, PAGE was performed for 2 h 30 min at constant voltage of 30 mA per gel in Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad, Hercules, CA, USA).
The eBlot transfer system (Genscript, Piscataway Township, NJ, USA) was used for protein transfer on NC membranes. After blocking with NET-G buffer for 1 h, the membranes were incubated with LC3B antibody (Abcam) at +4 °C overnight, washed, and incubated with the HRP-labeled anti-rabbit antibody (GE Healthcare, Chicago, IL, USA) for 1 h at RT. For visualization, Lumi-Light ECL (Roche) and the LAS3000 imager (Fujifilm, Minato, Tokyo, Japan) were used. Subsequently, membranes were washed, blocked with NET-G, and incubated with mouse β-actin antibody (Sigma-Aldrich) at +4 °C overnight. As the secondary antibody, HRP-labeled anti-mouse antibody (GE Healthcare) was used. β-actin was visualized with Lumi-Light ECL in LAS3000.

The strains used in this study were obtained from Yakult Culture Collection (Tokyo, Japan) and Japan Collection of Microorganisms (Ibaraki, Japan). Bifidobacterial strains were routinely cultured at 37 °C in an anaerobic chamber (Coy Laboratory, Grass Lake, MI, USA) with 88% N₂, 5% CO₂ and 7% H₂, using GAM Broth (Nissui Pharma, Japan) supplemented with 0.5% lactose and 0.5% glucose. The carbohydrate profile was evaluated at 37 °C in modified PY medium (100 mM PIPES, pH 6.7, 5 g/L peptone, 5 g/L BBL trypticase peptone, 10 g/L Bacto yeast extract, 8 mg/L CaCl₂, 19.2 mg/L MgSO₄·7H₂O, 40 mg/L K₂HPO₄, 40 mg/L KH₂PO₄, 0.4 g/L NaHCO₃, 80 mg/L NaCl, 4.9 mg/L hemin, 0.5 g/L L-cysteine hydrochloride, 100 ng/L vitamin K1 and 0.1% lactose) supplemented with filter-sterilised 2′-FL (Advanced Protein Technologies, Korea), fucose or lactose. Growth was monitored by measuring optical density at 600 nm every 0.5 h in an anaerobic chamber (Coy Laboratory, MI) using a PowerWave 340 plate reader (BioTek, VT, USA).