Notch Signaling Pathway Assay

S2 cells in 12-well dishes were transfected when approximately 50% confluent with combinations of pMT-Notch, pMT-DxV5, pMT-Pyd, NRE:Firefly (gift from S. Bray) and Actin:Renilla (gift from G.Merdes). After 24 h, when cells reached 100% confluence, they were re-suspended and seeded into white 96-well plates (Nunc #136101) and CuSO₄ was added after a further 24 h. 24 h after induction, luciferase activity was assayed with Dual-Glo Luciferase (Promega), quantified by a luminometer (Berthold), and Firefly/Renilla ratio was calculated for triplicate samples. For cell-density experiments, cells were re-seeded into white 96-well plates as above (high cell density) or diluted 1/10 with culture medium before reseeding (low cell density). For ligand-induced signalling assays, Notch expressing cells were layered on top of fixed Delta expressing S2 cells (S2-Mt-Dl; DGRC) according to the previously described protocol [4 (link)]. Experiments were repeated a minimum of three times.