Lb 940 multimode microplate reader

Cells were lysed in luciferase lysis buffer (1% (v/v) Triton X-100, 10% (v/v) glycerol, 25 mM glycylglycine (pH 7.8), 15 mM MgSO₄, 4 mM EGTA and 1 mM dithiothreitol). For dual luciferase measurement of Firefly and Renilla luciferase in 384- or 96-well microplates, cells were washed once with PBS, lysed directly on the plate in 20 µl (384-well plates) or 30 µl (96-well plates) luciferase lysis buffer per well and frozen at −80°C. Shortly before measurement lysates were allowed to thaw at RT for 30 to 60 min. Luciferase assay buffer (25 mM glycylglycin (pH 7.8), 15 mM K₂PO₄, (pH 7.8), 15 mM MgSO₄, 4 mM EGTA, 1 mM DTT and 2 mM ATP), supplemented with 70 µM D-luciferin (P.J.K., Kleinblittersdorf, Germany), was added to each well using a Multidrop 384 dispenser (Thermo-Fisher, Martinsried, Germany), and plates were incubated for 5 min at RT in the dark. Firefly luciferase activity was measured for 0.1 sec in a Mithras LB940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany). After addition of luciferase assay buffer, supplemented with 7.14 µM coelenterazine (P.J.K.), Renilla luciferase activity was measured for 0.5 sec using a 475 nm filter in the same multiwell reader.

Luciferase assays were performed in a 384-well format using white, flat-bottom polystyrene plates (781073; Greiner Bio-One, Frickenhausen, Germany) with at least seven technical replicates per biological replicate. On day 1, ∼2500 HEK293T cells in 50 µl culture medium were reverse transfected using 5 µl of 0.2 µM (Dharmacon) or 0.05 µM (Ambion) siRNA and 0.1 µl RNAiMAX in 10 µl serum-free RPMI 1640 medium per well. The next day, cells were additionally transfected using 0.1 µl TranIT-LT1 with 1 ng NanoLuciferase–WNT3 and 5 ng firefly luciferase for normalisation in 10 µl serum-free RPMI 1640 medium per well. 48 h later, the plate was centrifuged for 2 min at 650 g, and 20 µl of the medium was transferred to a second plate to measure NanoLuciferase–WNT3 in the supernatant. NanoLuciferase–WNT3 activity in supernatant and cell lysates, was detected using the Promega Nano-Glo (N1130; Fitchburg, USA) system and a Mithras LB 940 Multimode Microplate Reader (Berthold Technologies; Bad Wildbad, Germany). Luminescence signals in the supernatant were normalised to NanoLuciferase signals and firefly luciferase signals in the cell lysates.