HCE cells were cultured in 24 well plates until confluent. Once confluent, cells were stimulated with or without A. castellanii (1×105 cells/ml) and Ultra-pure LPS (10 μg/ml) (InvivoGen, San Diego, CA, USA) for 24 hours. HEK-293 cells were exposed to A. castellanii in vitro. A. castellanii trophozoites (1×105/ml) were added to 24 well plates containing HEK-293 cells (1×106 cells/ml) for 24 hours. Cells cultured without the trophozoites served as a control. As a positive control, transfected HEK-293 cells were activated with 125 ng/ml poly (I:C) (polyinosinic:polycytidylic acid), a specific ligand for TLR3 (InvivoGen, San Diego, CA, USA); and 100 ng/ml ultrapure LPS, a specific ligand for TLR4/MD2 (InvivoGen, San Diego, CA, USA) for 24 hours as described previously [28] (link). HCORN cells were cultured in 24 well plates until confluent and then cells were stimulated with or without A. castellanii (1×105 cells/ml), A. culbertsoni (1×105 cells/ml), A. astronyxis (1×105 cells/ml), A. castellanii Neff (1×105 cells/ml) and Ultra-pure LPS (10 μg/ml) (InvivoGen, San Diego, CA, USA) for 24 hours.
Cells and supernatants were collected by centrifugation at 2000×g for 10 min at 4°C and analyzed for the expression of TLR1, TLR2, TLR3, TLR4, TLR9, IL-8, CXCL2 and GAPDH genes by RT-PCR. IL-8 and CXCL2 production in the supernatants was determined by ELISA.
Cells and supernatants were collected by centrifugation at 2000×g for 10 min at 4°C and analyzed for the expression of TLR1, TLR2, TLR3, TLR4, TLR9, IL-8, CXCL2 and GAPDH genes by RT-PCR. IL-8 and CXCL2 production in the supernatants was determined by ELISA.