Model benchmark

Calu-3 cells were seeded (27,000 cells/cm² in 100 µl of DMEM-10% FBS medium) in 96 well-plates and grown to confluence. The cells were then incubated with 100 μg/ml CSE, 100 μg/ml CSE plus 10 μM IKK-2 inhibitor, 100 μg/ml CSE plus 5 mM NAC or the vehicle for 24 h. IL-6 and IL-8 were measured from supernatants using the human IL-6 and IL-8 ELISA set (BD OptEIA™ - Human IL-8 ELISA Set, and BD OptEIA™-Human IL-6 ELISA Set, BD Biosciences, San Diego, CA). Measurements were performed using a microplate reader according to manufacturer´s instructions (model Benchmark, Bio-Rad).

Enzyme activities were measured for three different samples: whole, lysed, and disrupted cells. Whole cell suspension was prepared as previously described (Park et al. 2012 (link)). Cell lysis step was carried out to extract microbial enzyme from the whole cell using lysis solution as described in Ku et al. (2011) (link). Disrupted cell suspension was prepared by the cell sonicator set at 45 amplifications for 3 min at 4 °C. For the enzyme reaction, 5 mM of ρ-nitrophenyl-β-D-glucopyranoside (pNPP), and ρ-nitrophenyl-β-D-glucofuranoside (pNPF) (Sigma, St. Louis, Mo., U.S.A.) were used. The released pNP was measured at 405 nm (Model Benchmark, Bio-Rad, Japan) after enzyme reaction at 37 °C. Enzyme activity was evaluated using the following equation: $$ln\left[A\right]/{\left[A\right]}_{\text{M}}=-kt$$ in which A = enzyme activity of BL47 cultured at modified MRS at time t; A_M = enzyme activity of BL47 cultured at normal MRS; k = rate constant; and t = time (min) (Ximenes et al. 2011 (link)).