Human amphiregulin duoset elisa

Cells were prepared in the same way as for RNA extraction. Conditioned medium was collected 24 h after treatment. HBEGF, AREG and EREG secretion was measured by ELISA (Human HB-EGF DuoSet ELISA, R&D Systems; Human Amphiregulin DuoSet ELISA, R&D Systems; Human Epiregulin ELISA Kit, Abcam) according to manufacturer’s instructions.

IL-9, EGFR, AREG, and HIF-1α levels were measured by commercial enzyme-linked immunosorbent assay (ELISA) kits purchased from R&D Systems (Minneapolis, MN, USA), including Human IL-9 DuoSet ELISA (Catalog Number: DY209-05), Human EGFR DuoSet ELISA (Catalog Number: DY231), Human Amphiregulin DuoSet ELISA (Catalog Number: DY262), and Human/Mouse Total HIF-1 alpha/HIF1A DuoSet IC ELISA (Catalog Number: DYC1935-2). Briefly, 100 µL of samples or standards were added to the wells of plates and incubated for 2 h at room temperature. The plates were washed five times. One hundred microliters of the detection antibodies were added to each well and incubated for 2 h at room temperature. The plates were washed five times. One hundred microliters of streptavidin-horseradish peroxidases were added to each well and incubated at room temperature for 20 min. The plates were washed five times. One hundred microliters of substrate solutions were added to each well and incubated at room temperature for 20 min. Fifty microliters of stop solutions were added to each well. The optical density of each well was determined using a microplate reader set to 450 nm.

Cell lines were treated and harvested as described for apoptosis assay. Additionally, Igrov-1 cells were treated with either 100 nM PMA (Sigma-Aldrich) or 200 nM of the anti-ADAM17 IgG antibody D1(A12) [30 (link)] or the equivalent amount of normal human IgG Control (R&D Systems, #1-001-A).
To investigate ADAM17 activity, AREG-release into culture supernatants was measured by human Amphiregulin Duoset ELISA (R&D Systems, DY262) and human TNFR1 was quantified by Duoset ELISA (R&D Systems, DY225) by detecting the OD at 450 nm using a microplate-reader (Infinite 200, Tecan). ELISA results were normalized to the total protein amount of cell lysates, which were quantified by BioRad Dc Protein Assay (#500-0112), using MS Excel (2010).