Human il 6 immunoassay

Conditioned media and cells were collected to examine the expression of human IL‐6 and EREG after exposure to IH for the indicated times (Fig. 1B,C, 2C, and 4B). IL‐6 and EREG were detected by a human IL‐6 immunoassay (R&D Systems, Minneapolis, MN, USA) and human EREG immunoassay (LifeSpan BioSciences, Inc., Seattle, WA, USA), respectively, according to the manufacturer's instructions. Briefly, cell lysates were prepared by repeats of freeze and thaw, and the protein assay was performed using coomassie brilliant blue solution. Conditioned media or cell lysates (10 μg of total protein) were added into a 96‐well plate, which was precoated with a monoclonal antibody specific for human IL‐6 or EREG. After washing away any unbound materials, an enzyme‐linked antibody specific for human IL‐6 or EREG was added to the wells. Following washing to remove any unbound antibody/enzyme reagent, a substrate solution was added to the wells. The intensity of the light emitted was measured by a microplate luminometer (POWERSCAN® HT; BioTek Instruments, Inc., Winooski, VT, USA).