Nahco3

RAW-Blue, HEK-Blue-hTLR4, HEK-Blue-hNOD2, and control HEK-Blue-Null2 cells were obtained from Invivogen (San Diego, CA, USA) and maintained in Dulbecco‘s Modified Eagle’s medium (DMEM) medium (GE Healthcare, Chicago, IL, USA) supplemented with 10% fetal calf serum (Thermo Scientific, Waltham, MA, USA), 50 U/mL penicillin, 50 μg/mL streptomycin, 2 mM glutamine, and 0.1 M NaHCO₃ (all PanEco, Moscow, Russia) at 37 °C with 5% CO₂.

Reporter cells (RAW-NF-κB-Luc, RAW-NFAT-Luc, and RAW-AP-1-Luc) were cultured in DMEM medium (GE Healthcare, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA), 50 U/mL penicillin, 50 μg/mL streptomycin, 2 mM glutamine, and 0.1 M NaHCO₃ (all PanEco, Russia) at 37°C with 5% CO₂. We used the protocol described in our previous work [16 (link)]; namely, experiment cells were seeded at 1 × 10⁵ cells/well in 96-well plates (100 μL/well). The next day, C-type lectin receptor agonists were added to the wells to a final concentration of 20 μg/mL, 4 μg/mL, and 1 μg/mL. Eight hours later, to detect luciferase activity, 100 μL of Bright-Glo Luciferase Assay Buffer containing luciferin substrate (Promega, USA) was added to each well. Luminescence was measured in relative units using a Synergy H4 Hybrid Reader (BioTek, Germany).