2.6. IL-8 ELISA assay
Quantitation of pro-inflammatory cytokine interlukin-8 (IL-8) was performed using Duoset ELISA kit (R&D Systems, USA, DY208). Caco-2 and hBMECs were cultured in GBA chip and 96 well. Each well used for culturing contained 0.3 ml of medium. Media obtained from the chip and wells with Caco-2 were diluted to 1/3 concentration while those from wells with hBMECs were diluted to 1/100 concentration. The assay followed the user manual. Briefly, 96 well used for the assay was pre-coated with capture antibody. Then, sample media were added into each pre-coated well and incubated. Sample was removed from each well, and then detection antibodies are added and incubated. Next, the antibodies were removed and streptavidin-HRP was added. After incubation avoiding direct light, streptavidin-HRP was removed, extract solution was added and incubated. Finally, stop solution was added to stop further reaction and absorbance of each well was measured using an UV-VIS spectrometer (Multiskan GO; Thermofisher, USA, N10588).