HT-29 and Caco-2 Reporter Cell Lines for Functional Studies of Nuclear Factor Kappa B Activation

Figure 7

Caco-2-NF-κB-hrGFP C3 and HT-29-NF-κB-hrGFP E5 and F6 clones showed different NF-κB activation (a) and IL-8 production (b) in response to LAB. Caco-2-NF-κB-hrGFP C3 cells were seeded at 1 × 10⁵ cell/well, while HT-29-NF-κB-hrGFP E5 and F6 clones were seeded at 5 × 10⁴ cell/well, cultured ON, and treated for 2 h with L. reuteri ATCC 23272 or L. plantarum ATCC 8014. Then, gentamycin and TNF-α (1 ng/mL) were added; 48 h or 18–24 h later (for Caco-2-NF-κB-hrGFP C3 and HT-29-NF-κB-hrGFP E5 and F6, resp.), cells were trypsinized and NF-κB activation (measured by the % GFP⁺ cells) was analyzed by flow cytometry. Cell viability was over 90% for all tested conditions. Data was normalized against TNF-α control (considered as 100%). IL-8 quantification was performed in the harvested cell culture supernatant by flow cytometry. Results were expressed as the mean ± SD of triplicates of a representative experiment. using One-Way ANOVA with Dunnett’s posttest.

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