Hek blue hdectin 1b cells

HEK-Blue™ hDectin-1b Cells (SEAP reporter cells) (Invivogen, San Diego, CA, USA) were plated in 96-well plates at 4 × 10⁵ cells/well in 0.2 mL/well of HEK-Blue™ medium (Invivogen, San Diego, CA, USA) and incubated overnight. MeOH was used as solvent to dissolve compounds and by performing the coating of the plate with 0.05 mL of solution of lipids at indicated concentration. Reporter cells activity was assessed after overnight incubation as quantification of secreted alkaline phosphatase (SEAP) released in the medium by reading the optical density (OD) at 655 nm. Zymosan was used as positive control under the same conditions at concentration of 100 µg/mL according to manufacturer’s instructions. Quantitative data (ng/mL) were obtained by a standard curve for the SEAP protein.

Hek-Blue hDectin-1b cells (InvivoGen) were grown in DMEM medium with 4.5 g/L glucose (Sigma-Aldrich), 10% heat inactivated Fetal Bovine Serum Premium (FBS) (Biowest), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (all from Sigma-Aldrich), 100 μg/mL Normocin™ and 1 µg/mL puromycin (both from InvivoGen) in vented T75 flasks. When cells reached 80% confluency, they were re-seeded at 5 × 10⁴ cells/well in flat-bottom 96 well-culture plates and stimulated with 10, 50 and 100 µg/mL of WGP^®-Soluble, WGP^®-Dispersible, or Zymosan in HEK-Blue™ Detection medium for 16 h. Substrate hydrolysis by secreted alkaline phosphatase (SEAP), upon activation of the receptor, was assessed at 620-655 nm according to manufacturer’s instructions in a BioteK™ µQuant Microplate Reader using Biotek™ Gen5™ Data Collection and Analysis Software (Thermo Fisher Scientific, Waltham, MA, USA).