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).