Pcmv ecfp eyfp

HEK293T cells were seeded in 6-well plates (3 × 10⁵ cells/well) and incubated overnight in DMEM with 5% FBS without antibiotics. On the next day, cells were transiently transfected with 0.5 µg of plasmid mixture of pcDNA3-hTLR4-YFP and pcDNA3-hTLR2-CFP (Addgene) or transfected with each plasmid (molar ratio 1:1) using Metafectene Pro (Biontex), according to the manufacturer’s protocol. Cells were also transfected with the plasmid pCMV-ECFP-EYFP (Addgene) that expresses the tandem CFP : YFP construct, which served as a positive control for FRET. A day after transfection, cells were cultured in 8-well glass bottom chambers and incubated overnight in phenol red-free DMEM with 5% FBS and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The following day, cells were stimulated with O. intermedium LPS for 30 min. FRET between TLR2 and TLR4 proteins was calculated by measuring sensitized emission fluorescence of CFP-YFP pair using NIS Elements 4.40 software on the Nikon Eclipse Ti-E confocal microscope. FRET image acquisition and processing details are provided in the Supplementary Material.