2.4.3. ELISA-Based Quantification of Substrates in Supernatants of 2D Cell Cultures and 3D Spheroids after Drug Treatment

Cell culture supernatants of 2D or 3D cell cultures 48 h after treatment were collected to investigate substrate release. The concentration of HB-EGF was measured as a surrogate for ADAM17 activity. Therefore, the Human HB-EGF DuoSet ELISA (R&D Systems, #DY259B) was used according to the manufacturer’s instructions in NUNC-IMMUNO plates (Thermo Scientific, #442404, Waltham, MA, USA). Using a microplate reader (Infinite 200, Tecan), the optical density (OD) was measured at 450 nm, and HB-EGF concentrations were calculated using MS Excel (2010). The means of three independent experiments + standard error of the mean (SEM) were determined using GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA).