Mithras lb 940 multimode plate reader

For Dual-Luciferase assays, C2C12 cells were transfected in 96-well plates with inducible BRE-luciferase or (CAGA)₁₂-luciferase reporter gene constructs together with constitutively expressed Renilla luciferase (pRLTK-luc) (41 (link), 42 (link)). After 16 h, cells were starved for 2 h and stimulated for 6 h with ligand in the presence or absence of kinase inhibitors as described at the indicated concentrations. Luciferase activity was quantified using the Dual-Luciferase reporter gene assay in a Mithras LB940 multimode plate reader (Berthold Technologies).
For Myg-luciferase assays, C2C12 cells were transfected in 96-well plates with the myogenin-luciferase construct (43 (link)). At confluence, cells were pretreated with inhibitors for 30 min before stimulation with ligand as indicated or DMSO vehicle control in differentiation medium (DMEM plus 2% horse serum). After 3 days of differentiation, cells were lysed, and firefly luciferase activity was measured as described.

After plating, cells were left to adhere overnight, before stimulation with a range of concentrations (0 to 10 mM) of the ammonia donor, NH₄Cl, in the absence or presence of 100 nM CNP, in DMEM supplemented with 1% (v/v) FCS and 1% (v/v) antimycotic/antimicrobial, to reduce the basal rate of proliferation. At the indicated time points (24, 48 and 72 h), cells were washed twice with PBS, before being fixed with 4% (w/v) paraformaldehyde-containing PBS and stored at 4 °C prior to staining with 0.075% (w/v) crystal violet solution for 15 min, followed by subsequent washing in water and drying overnight. The stained cells were dissolved in 10% (v/v) acetic acid and left for 30 min, before measuring the optical density at 595 nm using a Mithras LB940 Multimode Plate reader (Berthold, Harpenden, UK). Experiments using GPNT cells also included treatments in the absence and presence of 1 mM SNP.