To measure the activity of secreted human placental alkaline phosphatase (SEAP), the increase of absorbance at 405 nm due to hydrolysis of para-nitrophenyl phosphate (pNPP) in the cell culture supernatant was followed (1 reading per minute for 25 minutes). To this end, 60-100 μL of supernatant was collected from each well and heat-inactivated for 30 minutes at 65 °C. 180 μL of assay reagent containing 100 μL 2x SEAP buffer (20 mM homoarginine, 1 mM MgCl2, 21% (v/v) diethanolamine, pH 9.8), 20 μL of pNPP substrate solution (20 mM, in 2x SEAP buffer) and 60 μL of water were added to 20 μL of heat-inactivated supernatant. The absorbance at 405 nm at 37 °C was monitored with a Tecan M1000 multiplate reader (Tecan AG) and used to calculate SEAP activity.
Cell culture supernatant was also used to determine the activity of secreted nano-luciferase with the Nano-Glo® Luciferase Assay System (N1110, Promega). To this end, 7.5 μL of cell culture supernatant was mixed with 7.5 μL buffer/substrate mix per well in a 384-well black-well plate. Luminescence of the reporter was measured using a Tecan M1000 multiplate reader (Tecan AG).
Cell culture supernatant was also used to determine the activity of secreted nano-luciferase with the Nano-Glo® Luciferase Assay System (N1110, Promega). To this end, 7.5 μL of cell culture supernatant was mixed with 7.5 μL buffer/substrate mix per well in a 384-well black-well plate. Luminescence of the reporter was measured using a Tecan M1000 multiplate reader (Tecan AG).