Hek blue reporter cell lines

TLR-2 and TLR-4 agonists were measured using HEK-Blue reporter cell lines according to the manufacturer’s instructions (InvivoGen, San Diego, CA, USA). Cells were exposed to the supernatants of caecal content and the protocol previously described was followed [31 ].

HEK-Blue™ reporter cell lines (InvivoGen) transfected with human TLR2, human Dectin1a, human TLR4/CD14/MD2 or non-transfected (null-1) were cultured and maintained using DMEM GlutaMAX™ containing 10% FBS (Sigma-Aldrich), Normocin (100 μg/mL) and HEK-Blue™ selection antibiotics. Experiments were carried out according to the manufacturer’s instructions. Briefly, I. obliquus polysaccharides (20 µL) at various concentrations were added to wells in 96-well plates (Costar). Then, the reporter cells were gently washed with warm PBS before suspended in HEK-Blue™ Secreted Embryonic Alkaline Phosphatase (SEAP) detection medium. Finally, 180 µL 5 × 10⁴ reporter cells were added per well containing polysaccharides. After incubation for 16 h (37 °C, 5% CO₂), SEAP was detected colorimetrically at A₆₃₅. The experiments were carried out at least three times.

Toll like receptor (TLR) 2 and TLR4 agonists were measured using Hek-Blue reporter cell lines according to manufacturer instructions (InvivoGen, San Diego, CA, USA). Cells were maintained in RPMI medium with ultra-low endotoxin FBS (Biosera, Nuaille, France) with appropriate antibiotics (Normocyn, Zeocyn and/or HekBlue selection, InvivoGen, San Diego, CA, USA). We resuspended fecal material or Spirulina in LAL reagent water (Lonza, Walkersville, MD, USA) to a final concentration of 100 mg/mL and homogenized for 4 min using a tissue lyzer without the addition of beads to avoid bacteria disruption. We then centrifuged the samples at 8000× g for 2 min, serially diluted the resulting supernatant, heat them at 56 °C for 45 min, and applied them to cells. Purified Escherichia coli LPS (Sigma, St. Louis, MO, USA) and FSL-1 (InvivoGen, San Diego, CA, USA) were used for standard curve determination using HEK-Blue mTLR4 and HEK-Blue mTLR2 cells, respectively. After 21 h of stimulation, we applied cell culture supernatant to QUANTI-Blue medium (InvivoGen, San Diego, CA, USA) and measured the alkaline phosphatase activity at 620 nm after 3 h. A control cell line (HEK-Blue Null1 cells) was included to remove unspecific signals from the fecal slurry.