Mithras lb940 reader

BRET proximity assays were performed as described previously (23 (link)). Briefly, HEK-293T cells were transfected using a constant amount of plasmid DNA but various ratios of plasmids encoding the fusion protein partners. A control corresponding to mock-transfected cells was included in order to subtract raw basal luminescence or fluorescence from the data. Expression of mVenus fusion proteins was estimated by measuring fluorescence at 535 nm following excitation at 485 nm. Expression of RLuc fusion proteins was estimated by measuring the luminescence of the cells after incubation with 5 μM coelenterazine H (Promega). In parallel, BRET¹ between hRLuc8 and mVenus was measured 5 min after addition of 5 μM coelenterazine H (Promega). BRET¹ readings were collected using a Mithras LB940 reader (Berthold). The BRET¹ signal was calculated as the ratio of emission of mVenus (510–590 nm) to hRLuc8 (440–500 nm)—Cf, where Cf corresponds to the ratio of emission (510–590 nm) to (440–500 nm) for the hRLuc construct expressed alone in the same experiment.

Proliferation was assessed using the Click-iT EdU Proliferation Assay for Microplates (C10499, Invitrogen). Briefly, cells were plated at 48% confluency in 96 well black microplate (CLS3603, Corning, MERCK), labeled with 10 µM EdU for 24 h, then fixed and clicked according to the manufacturer’s instructions. Fluorescence was analyzed with a Mithras LB 940 reader (Berthold technologies, Bad Wildbad, Germany) and MikroWin 2010 software (Berthold technologies) using excitation filter 560 nm and emission filter 590 nm.