HEK293 cell activation assays: Dual Luciferase Reporter Assay and AlphaLISA Assay

HEK293 cells were seeded into 96-well Costar plates (Corning, NY, USA) with 10% FBS in DMEM, at 3 × 10⁴ cells/well, and incubated overnight in a humidified atmosphere (5% CO₂) at 37°C. The next morning, cells were co-transfected for 4 h with pEFBOS- (wt or mutant, h or m) MD-2-FLAG-His and pUNO- (h or m) TLR4-HA together with NF-κB-dependent luciferase and constitutive Renilla reporter plasmids using Lipofectamine 2000 (Invitrogen, Life Technologies Waltham, MA, USA). After 4 h, medium was removed and replaced with DMEM + 10% FBS. The following day, cells were incubated with S-LPS for 16 h, as indicated. In selected experiments, HEK293 cells were seeded into 96-well plates (3 × 10⁴ cells/well) and separately transfected either with mMD-2 (wt or mutant) or with TLR4 (h or m) together with NF-κB-dependent luciferase and constitutive Renilla reporter plasmids. After 16 h, aliquots of the conditioned medium of HEK293 cells containing sMD-2 were added to HEK293TLR4 cells where the supernatants have been removed. The cells were then activated with lipid A for 16h. In a different approach, HEK293 cells were seeded into 12-well plates (3 × 10⁵ cells/well) and transfected either with mMD-2 (wt or mutant) or with TLR4 (h or m) together with NF-κB-dependent luciferase and constitutive Renilla reporter plasmids. After 16 h, the cells were resuspended in fresh medium containing 10% serum, joined in 1:1 ratio and re-seeded together in 96-well plates to yield co-cultures of cells separately expressing MD-2 or TLR4. The following day, the cells were incubated in the presence of S-LPS or lipid A for 16 h. After the activation with S-LPS or lipid A, the supernatants were harvested and the cells were lysed in 1 x reporter assay lysis buffer (Promega, Fitchburg, WI, USA) and analyzed for reporter gene activities using a dual-luciferase reporter assay system on a Mithras LB940 luminometer. Relative luciferase activity (RLA) was calculated by normalizing each sample’s luciferase activity for constitutive Renilla activity measured within the same sample. When plotting data the value of the unstimulated sample with wt hMD-2 was set to 1 and other values were adjusted accordingly. In the supernatants of HEK293 cells, IL-8 concentrations were determined with AlphaLISA IL-8 Immunoassay Research kit (Perkin Elmer), according to the manufacturer’s instructions.