Hek blue clr selection

HEK-Blue hDectin-1a cells (InvivoGen; catalog hkb-hdect1a) were maintained in growth medium containing DMEM (4.5 g/L glucose), 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL Normocin (InvivoGen; catalog ant-nr-05), 2 mM l-glutamine, 1 μg/mL puromycin (InvivoGen; catalog ant-pr-1), and 1× HEK-Blue CLR Selection (InvivoGen; catalog hb-csm). This cell line expresses the Dectin-1a isoform and genes involved in the Dectin-1/NF-κB/SEAP signaling pathway. On the assay day, 180 μL/well of cells at a concentration of 2.8 × 10⁵ cells/mL in HEK-Blue Detection media (InvivoGen; catalog hb-det2) were plated in a 96-well tissue culture plate. Plasma samples (20 μL) were added to each well with and without piceatannol (250 nM). The plates were then incubated at 37°C and 5% CO₂ for 24 hours. Levels of SEAP were monitored and measured spectrophotometrically at 620 nm. As controls, 20 μL of water, piceatannol (250 nM), 2 μL of DMSO (piceatannol solvent), 20 μL of 10 μg/mL β-glucan peptides (InvivoGen; catalog tlrl-bgp), or 20 μL of β-glucan peptides + piceatannol (250 nM) were added in separated wells.

HEK-Blue or RAW-Blue NFκB-SEAP reporter cells (Invivogen) were cultured using Puromycin and HEK-Blue™ CLR Selection (Invivogen) as selective antibiotics and DMEM (4.5 g/L glucose), 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin and 2 mM L-glutamine. Reporter cell assays were carried out by seeding at 50,000 cells per well in 180 μL in a 96 well flat-bottomed plate and incubating the cells with 20 μL of the indicated agonists overnight. SEAP activity was measured using QUANTI-Blue (Invivogen) according to the manufacturer’s instructions. Briefly, 20 μL of supernatant was added to 180 μL of QUANTI-Blue solution and incubated at 37°C for 15 minutes. Optical density at 620 nm was then measured using a plate reader.