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).
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).
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