ELISA Assay for IL-6 in HNEC-ALI

Interleukin-6 (IL-6) levels produced by HNEC-ALI cultures were measured with enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, Franklin Lakes, NJ, USA) after application of 4 mM Der p 1, 4 mM Der p 2 for 6 h. PBS was used as negative control and 10 μg/ml poly (I:C) LMW (Invivogen, San Diego, CA, USA) was used as positive control. The medium in the basolateral chamber was harvested after completion of the TEER experiment and stored at −20°C. Amounts of IL-6 in the medium were determined according to the manufacturers' protocols. All samples were measured in duplicate. The optical density (OD) was measured at 450 nm and IL-6 concentration was determined using the standard curve prepared for individual assay (21 (link), 23 (link), 24 (link)).