Muramyl dipeptide

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.

For the preparation of heat-killed bacteria, L. monocytogenes (10403S), E. coli (ATCC 25922), S. aureus (ATCC 29213), S. typhimurium (ATCC 14028) and S. iniae (ATCC 29178) were inoculated from glycerol stocks or blood agar plates and cultured in brain heart broth (BHB) (Sigma-Aldrich, MO, USA) at 37°C until the OD600 nm reached 0.9-1.0. Bacterial suspensions were plated on LB agar plates to verify bacterial concentrations. To heat-kill bacteria, the bacterial suspensions were autoclaved in BHB at 120°C for 20 min and the sterility was confirmed by plating on LB plates after autoclaving. Injection doses of heat-killed bacteria for adult zebrafish were 0.5×10⁷-1×10⁷ cfu. Injection doses for other priming agents were 13.5 µg/fish for LPS (Sigma-Aldrich, MO, USA), paclitaxel (Sigma-Aldrich, MO, USA) and zymosan (Sigma-Aldrich, MO, USA), and 4.5 µg/fish for muramyl-dipeptide (Sigma-Aldrich, MO, USA). Priming i.p. injections (5 µl) were injected with an Omnican 100 30 G insulin needle (Braun, Melsungen, Germany) under 0.02% 3-aminobenzoic acid ethyl ester (pH 7.0) anesthesia.