Costar white 96 well plates

HEK293 cells were harvested from an actively growing culture and plated onto CoStar White 96-well plates (Corning) at 2.2 × 10⁴ cells per well (0.1 ml). After 24 h at 50% confluence, the cells were transiently transfected with plasmids expressing wt TLR9 (20 ng DNA per well) or pcDNA3 (Invitrogen), UNC93B1 (5 ng DNA per well), ELAM1-luciferase reporter plasmid (50 ng DNA per well), and phRL-TK (5 ng DNA per well, unless stated otherwise) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). After 24 h, the culture medium was replaced with fresh medium, and the cells were stimulated with TLR9 agonists for 18 h. The cells were lysed in passive lysis buffer (Promega) and analyzed for reporter gene activities using a dual-luciferase reporter assay. Error bars represent the s.d. obtained from at least three biological replicates.

HEK293 cells were plated onto CoStar White 96-well plates (Corning) at 2.2×10⁴ cells/well. After 24 h, the cells were transfected with a following plasmids: pIFN-β-luciferase (40 ng DNA/well) or ELAM1-luciferase reporter plasmid (40 ng DNA/well), pUNO-hTLR3-HA (20 ng DNA/well), pUNO-hTLR9-HA (20 ng DNA/well) or TLR3-TLR9 chimeric constructs (20 ng DNA/well), pUNO1-hUNC93B1 (1 ng DNA/well) and phRL-TK (5 ng DNA/well). Empty vector pcDNA3 (20 ng DNA/well) was used as a negative control. Plasmids were transfected using Lipofectamine 2000 reagent according to manufacturer's instructions (Invitrogen). 24 h post transfection, the cells were stimulated with TLR ligands: poly(I:C) (10 μg/ml) and ODN10104 (10 μg/ml). 18 h after treatment, the cells were lysed in Passive Lysis Buffer (Promega). The expression of the firefly and Renilla luciferase reporter gene was analyzed using the dual luciferase assay. Luminescence was quantified using the plate reader OrionII (Berthold Technologies). The relative luciferase expression (relative luciferase unit - RLU) for each sample was calculated by normalizing firefly luciferase activity for constitutive Renilla luciferase activity measured within the same sample.