Hek blue htlr4

Toll-like receptor screening was conducted by seeding HEK-Blue hTLR2-TLR1, HEK-Blue hTLR2-TLR6, HEK-Blue hTLR2, HEK-Blue hTLR3, HEK-Blue hTLR4, HEK-Blue hTLR5, HEK-Blue hTLR7, HEK-Blue hTLR8, and HEK-Blue hTLR9 (InvivoGen) into 96-well plates. Phages OMKO1, LPS-5, PSA04, and PSA34 were added at a concentration of 1 × 10¹⁰ PFU/ml in PBS supplemented with 0.9 mM CaCl₂ and 0.5 mM MgCl₂ and incubated at 37°C for 24 h with 5% CO₂. At the end of the incubation, NF-κB activity was monitored by the production of secreted embryonic alkaline phosphatase (SEAP) through the hydrolysis of HEK-Blue (InvivoGen). Optical density was read at 650 nm using a SpectraMax340 PC plate reader (Molecular Devices).

For NFkB reporter assay, HEK-Blue™ hTLR4 (InvivoGen) was incubated with LPS (100ng/ml), LNFPIII (50μg/ml), Dextran carrier (50μg/ml) or nothing for 24 hrs at 37°C. Supernatant was collected and alkaline phosphatase levels were measured by adding the substrate as per manufacturer’s instruction.

Human type II alveolar epithelial cells (HAECs) isolated from human normal lung tissue were purchased from AcceGen (AcceGen Biotech, Fairfield, NJ, USA) and cultured in an HAEC medium kit according to the manufacturer’s instructions. The human embryonic kidney HEK293 cell line was kindly provided by Professor B. Friguet (Sorbonne Université, Paris, France) and grown in Minimum Essential Medium Eagle (MEM) (Merck, Darmstadt, Germany), supplemented with L-Alanyl-L-Glutamine (0.4 g/L) and 10% heat-inactivated fetal calf serum (FCS) (Gibco, Dublin, Ireland). HEK-BLUE-hTLR4 stably expressing human TLR4 and an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene was purchased from InvivoGen (San Diego, CA, USA) and grown in Dulbecco’s Modified Eagle Medium (DMEM)—high glucose (Merck), supplemented with 10% heat-inactivated FCS and antibiotic mixture (HEK-Blue^TM Selection) for the persistent expression of transgenes. The human monocytic leukemia cell line (THP-1) was kindly provided by Dr S. André (Sorbonne Université, Paris, France) and maintained in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. THP-1 was grown to a density of 500,000 cells/mL and used for experiments between passage numbers 5 and 10. All cells were cultured under a humidified atmosphere in a 95% air–5% CO2 incubator at 37 °C.

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.