Hek blue htlr5 cells

HEK-Blue™ hTLR5 cells (InvivoGen) were seeded in 96-well cell culture plates 24 h prior to stimulation at a density of 2.52 × 10⁴ cells/well in cell culture medium. Before adding the proteins, bacteria or infection supernatant samples, cells were washed once with sterile medium. From each sample and negative control, 20 µL were applied. In bacterial approaches, stationary cultures adjusted to 10⁸ CFU/mL were used. Protein samples were derived from the native source and used in final concentrations of 0.05–100 ng/mL for L. pneumophila FlaA and 0.1–5000 ng/mL for ProA. After adding 180 µL of HEK-Blue™ Detection medium (InvivoGen), the eukaryotic cells were incubated for 16 h at 37 °C and 5% CO₂. The cell line contains an SEAP (secreted embryonic alkaline phosphatase) reporter gene, which is induced by the transcription factors NF-κB and AP-1 in the downstream signaling process after receptor stimulation of hTLR5. SEAP activity was measured at OD₆₂₀.

HEK 293T cells (CRL-3216; ATCC, Manassas, VA, USA) and the mouse monocyte/macrophage cell line J774A.1 (TIB-67; ATCC, Manassas, VA, USA) were each cultured in Dulbecco’s Minimal Essential Media (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 10 mM HEPES, 100 U/mL penicillin and 100 μg/mL streptomycin (all from Corning, Tewksbury, MA, USA). The human monocyte cell line, THP-1 (TIB-202; ATCC, Manassas, VA, USA) was cultured in RPMI-1640 and the same supplements as well as 0.05 mM 2-mercaptoethanol (Sigma, St Louis, MO, USA). HEK-Blue-hTLR5 cells (Invivogen, San Diego, CA, USA) were cultured in Dulbecco’s Minimal Essential Media (DMEM) supplemented with 10% FBS, 10 mM HEPES, 50 U/mL penicillin, 50 μg/mL streptomycin, 30 μg/mL blasticidin and 100 μg/mL zeocin (the latter two reagents from Invivogen, San Diego, CA, USA). All cells were cultured at 37 °C and 5% CO₂ in a humidified incubator.

Determination of TLR5 bioactivity with the putative Lawsonia flagellin was conducted using HEK-Blue™-hTLR5 cells (InvivoGen, San Diego, CA, USA). The cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), zeocin (50 μg/mL), and blasticidin (1 μg/mL). Cells (1 × 10⁶ cells per well) that had been incubated overnight in a 96-well plates were stimulated with the LFliC protein (10 μg) in duplicate for various time periods (0, 1, 3, 4, and 7 h). The expression of TLR5 on the stimulated cells was assessed by fluorescence-activated cell sorting (FACS) analysis using an anti-TLR5 monoclonal antibody (1 μg/mL; Anti-hTLR5-IgG2a, Thermo Fisher Scientific, Waltham, MA, USA) as previously described [16 (link)]. Furthermore, expression of IL-8 cytokine produced by signaling through TLR5 was measured in the stimulated cells by reverse transcription-polymerase chain reaction (RT-PCR). The primers for the IL-8 gene were obtained previously [17 (link)]. Briefly, 1 × 10⁶ HEK cells were treated with two concentrations of LFliC (10 and 100 ng/mL) in duplicate. The treated cells were incubated at 37 °C in a 5% CO₂ incubator for 24 h and the transcription level of IL-8 was determined by RT-PCR as described previously [18 (link)].