Rpmi culture media

To study the interaction of pectin with TLRs, we used the HEK-Blue™ TLR cell based assays from InvivoGen, Toulouse, France. These HEK-Blue™ cells express the soluble embryonic alkaline phosphatase (SEAP) gene, which can be quantified using Quanti-Blue (InvivoGen, Toulouse, France). The SEAP gene is under the control of the NF-κB/AP-1 responsive promoter. These HEK-Blue™ cells also co-express single TLR genes. Upon activation with TLR specific agonists, NF-κB is activated leading to SEAP expression. We used HEK-Blue™ hTLR4, hTLR5 cells (InvivoGen, Toulouse, France), and HEK-Blue™ Null1 TLR2 (developed by us) for studying the interaction of TLRs and pectins. HEK-Blue™ Null1 (InvivoGen, Toulouse, France) is the parental cell line for HEK-Blue™ TLR cells, expressing the SEAP gene, but not any TLR expression construct (34 (link)). Cell lines, antibiotics, and concentration of agonists used to activate TLR signaling are shown in Table 1. Mouse macrophage cells RAW264.7 (ATCC, VA, USA) were used to determine TLR2 inhibition using pectin.
HEK cells were cultured in DMEM culture media (Lonza, Basel, Switzerland), and RAW264.7 were cultured in RPMI culture media (Lonza, Basel, Switzerland) with 10% de-complemented fetal calf serum, 50 U/ml penicillin (Sigma, St. Louis, MO, USA), 50 µg/ml streptomycin (Sigma, St. Louis, MO, USA), and 100 µg/ml normocin (InvivoGen, Toulouse, France).

Human HCC cell lines SNU-423 (ATCC-CRL-2238) and SNU-182 (ATCC-CRL-2235), and human immortalized liver cell lines THLE-2 and THLE-3, were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA). HCC cells were grown in RPMI culture media (Lonza, Walkersville, MD, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Waltham, MA, USA). THLE2 and THLE3 cells were cultured in a BEGM Bullet kit (Lonza), which contains BEBM basal medium and supplements. The final growth medium consists of BEBM supplemented with 10% FCS, bovine pituitary gland extract, hydrocortisone, epidermal growth factor (EGF), insulin, triiodothyronine, transferrin, and retinoic acid. THLE cells require a special coating medium that consists of the following: RPMI1640 without glutamine supplemented with 0.01 mg mL⁻¹ bovine serum albumin, (heat shock fraction, Sigma, Burlington, MA, USA), 0.03 mg mL⁻¹ type I collagen from bovine skin (Sigma), and 0.01 mg mL⁻¹ fibronectin from human plasma (Sigma). All cell lines were maintained independently in the recommended medium at 37 °C and 5% CO₂.