Immunomodulation Assays using HT-29 Cells

HT-29 human epithelial cells were used for immunomodulation assays; either the parental lineage (HT-29, colon adenocarcinoma; ATCC HTB-38) or a lineage transfected with the secreted alkaline phosphatase (SEAP) reporter gene for NF-kB activation monitoring (HT-29/kb-seap-25) (Lakhdari et al., 2010 (link)). The reporter HT-29/kb-seap-25 cells were cultured in RPMI-Glutamine medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Corning), 1% non-essential amino acids, 1% sodium pyruvate, 1% HEPES buffer (Thermofisher Scientific), and 1% penicillin-streptomycin (Lonza) according to Lakhdari et al. (2010) (link). The parental HT-29 cells were cultured in high-glucose DMEM medium (Dominique Dutscher) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Do Carmo et al., 2017 (link)). For subcultures, cells were rinsed with DPBS (Thermofisher Scientific) and detached with a trypsin (0.05%) – EDTA (0.02%) solution (Sigma). Periodically, 100 μg/mL Zeocin (Invivogen) was applied to the HT-29/kb-seap-25 cell culture in order to maintain selective pressure on the cells containing the transfected plasmid.