Hek 293 htlr4 md2 cd14 cells

Stably transfected HEK 293 hTLR4/MD2/CD14 cells (InvivoGen, San Diego, CA, USA) were seeded into 96-well plates at the density of 3 × 10⁵ cells/ml. Cells were transfected with Firefly luciferase reporter constructs, pGL3.ELAM.tk, and Renilla luciferase reporter plasmid, pRLTK as published33 (link). HEK.293 hTLR4/MD2/CD14 were exposed to 100 ng/ml E. coli LPS (LPS-EB ultrapure, InvivoGen) or to 100 ng/ml TpF1. Cells were stimulated for 4 h and 18 h. Similarly, stably transfected HEK293 hTLR2 cells (InvivoGen) were exposed to 100 ng/ml TpF1 or to 1 μg/ml Pam3CSK4 (InvivoGen). Stimulation was carried out for 6 h and 18 h. NF-κB-dependent luciferase activity was measured at 4 h (for hTLR4/MD2/CD14) and 6 h (for hTLR2) using the Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WV, USA), as reported33 (link). IL-8 release was quantified at 18 h of stimulation by ELISA. Absence o bacterial contaminants in recombinant VEGF was tested by the manufacturer (Immunological Sciences).

The plasmids pNF-κB-luc (firefly luciferase), pEFIRES-MD2, phRG-TK (renilla luciferase), pcDNA3-CD14, and a pCMV vector containing TLR4 cDNA with an N-terminal FLAG tag (pCMV-TLR4-FLAG) were provided by N. Gay [43 (link)]. HEK293 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM: Gibco, Life Technologies Ltd.) supplemented with 4.5 g/l glucose, 10% foetal bovine serum (FBS: HyClone Laboratories Inc.), penicillin (50 U/ml) and streptomycin (50 μg/ml) at 37°C, 5% CO₂. The EAC lines OE33 (European Collection of Cell Cultures (ECACC)), SKGT4 (ECACC), OE19 (Sigma-Aldrich), OACP4C and OACM5.1 (both provided by W. Dinjens [44 (link)]) were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% FBS. JH-EsoAd1 cells were obtained from H. Alvarez [45 (link)] and grown in Minimal Essential Medium (MEM: Gibco) supplemented with 10% FBS. HEK293-hTLR4-MD2-CD14 cells (InvivoGen) were cultured in DMEM with 4.5 g/l glucose, 10% FBS, 50 U/ml penicillin, 50 μg/ml streptomycin and 100 μg/ml Normocin (InvivoGen). Selective antibiotic pressure was maintained with 10 μg/ml Blasticidin (InvivoGen) for hTLR4 and 50 μg/ml Hygrogold (InvivoGen) for MD2 and CD14 plasmids. FLO-1 cells (from D. Beer [46 (link)]) were cultured DMEM with 4.5 g/l glucose and 10% FBS.