Hek blue htlr4

HEK-Blue^TM hTLR2 and HEK-Blue^TM hTLR4 cells (InvivoGen, San Diego, CA, USA) were maintained in growth medium (DMEM, 4.5 g/L glucose, 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 ug/mL streptomycin, 100 ug/mL normocyn) with 1× HEK-Blue^TM Selection (InvivoGen). The parental cell lines HEK-Blue^TM Null1 and Null2 (InvivoGen) were used as controls for HEK-Blue^TM hTRL2 and HEK-Blue^TM hTLR4, respectively. Only cells with 20 passages or less were used. About 5 × 10⁴ cells/well were incubated with 0.5 µg/mL L. prolificans or S. boydii α-glucan for 18 h at 37 °C; 25 ng/mL Pam3CSK4 (TLR2 agonist) and 10 ng/mL LPS (TLR4 agonist) were used as control. After incubation, 20 µL of cell culture supernatant were incubated with 180 µL of QUANTI-Blue^TM solution (InvivoGen) at 37 °C for 4 h. Alkaline phosphatase activity (the reporter gene for HEK-Blue^TM hTLR4 and hTLR2) was measured by optical density (OD) measurement at 620–655 nm using a microplate reader. Polymyxin B, a cyclic polypeptide antibiotic, was used to eliminate the effects of endotoxin contamination in some experiments with HEK-Blue^TM hTLR4 cells.

HEK-Blue™ hTLR4, HEK-Blue™ hTLR2, and HEK-Blue™ Null2™ cells (InvivoGen, San Diego, CA, USA) were cultured at 37 °C in 5% CO₂ using Dulbecco’s minimal essential media (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Microgem, Naples, Italy), 1% glutamine (Himedia Einhausen, Germany), 1% penicillin/streptomycin (Himedia, Germany), and 100 µg/mL Normocin (InvivoGen). Plasmid selection in HEK-Blue™ hTLR4 and hTLR2 cells required the use of a mixture of selective antibiotics (HEK-Blue™ Selection) (InvivoGen), whereas HEK-Blue Null2™ cells required the use of 100 µg/mL Zeocin (InvivoGen).

HEK-Blue^TM hTLR4 and HEK-Blue^TM Null2^TM cells were purchased from InvivoGen, San Diego, CA, USA. The cells were cultured at 37 °C in 5% CO₂ in 25 or 75 cm² vented flasks using Dulbecco’s minimal essential media (DMEM) containing glutamine, heat-inactivated fetal bovine serum (FBS), penicillin/streptomycin, and normocin (InvivoGen). All culture media and plastics were purchased as endotoxin-free, and all glassware was de-pyrogenated by baking at 250 °C for 2 h. Selection of the plasmids in HEK-Blue hTLR4 cells required the use of HEK-Blue^TM (InvivoGen), and in HEK-Blue Null2 cells required the use of zeocin (InvivoGen). The cells were harvested for stimulation as non-confluent (50–70% of confluence) using Ca⁺- and Mg⁺-free Hank’s balanced salt solution (HBSS; Sigma). Cells were not centrifuged, as sufficient numbers were available for subculture in the dislodged cells. Clumps were eliminated by sedimentation at 100× g. Cell counts and viabilities were determined by hemocytometer and Trypan Blue exclusion. The cells were plated at 0.5–2.0 × 10⁵ viable cells per 100 μL per well on flat bottom 96-well plates (CellStar, Greiner Bio-One, Monroe, NC, USA).