Figure 2. Luciferase assay configurations.
A. The dual luciferase assay protocol is illustrated. Cells are lysed with a detection reagent containing luciferase substrates (1), FLuc luminescence is measured (2), the FLuc reaction is then stopped along with the addition of the coelenterazine substrate for RLuc (3), and luminescence is again measured (4). In this case total luminescence is measured using a clear filter; the luminescence emission spectra of FLuc and RLuc are also shown (CPS, counts per second measured on a luminometer). B. CBLuc two-color dual-luciferase assay protocol is shown in which cell lysis and detection are performed with a single reagent addition (1) and green luminescence and red luminescence are then measured sequentially on a microtiter plate reader (2) using the appropriate optical filters. The luminescence emission maximums are noted and the luminescence emission spectra for both green and red CBLuc are shown below. The dotted lines on the emission spectra represent the optical filters used in (Davis et al., 2007). C. A live cell kinetic assay is depicted using a secreted luciferase (GLuc) that uses coelenterazine as the substrate. Cell culture supernatant containing the secreted GLuc is removed at different times (1) and upon mixing with the coelenterazine substrate (2) the total luminescence for each time point is measured using a clear filter (3). Also shown is the emission spectra for GLuc relative to RLuc (spectra adapted from (Tannous et al., 2005). The GLuc wavelength emission maximum is similar to RLuc, but GLuc shows brighter luminescence.