An ELISA assay (Cell Signaling Technology 7276) containing members of key inflammatory signaling pathways, including NF-κB, SAPK/JNK, p38 MAPK, and STAT3, was used. Primary murine CECs were isolated as previously described (24 (link)) and normalized to 1.5 × 106/mL by hemocytometer and cultured for 4 h in the presence or absence of tomato extracts. Aqueous tomato extracts were added to a 2% final concentration (volume/volume ratio). The cultures were then stimulated with bacterial peptides (MAMPs); 1 µg/mL of peptidoglycan (S. aureus, Sigma), 1 µg/mL of muramyl dipeptide (Bachem), and 1 µg/mL of lipopolysaccharide (S. typhosa, Sigma) for 30 min, except the uninduced control. The assay was replicated three times. Stimulated cells were then lysed immediately following the protocol recommended by the manufacture. Protein concentrations were checked by Lowry protein assay (Bio Rad 500-0120), and samples were normalized to 700 µg/mL. Cell lysates were diluted 1:1 with sample dilutant (Cell Signaling Technology) and 200 µL of diluted lysates were used per well.
To determine if the ELISA results were significantly different, single-factor ANOVA analysis was performed.
To determine if the ELISA results were significantly different, single-factor ANOVA analysis was performed.