Furimazine (Promega), a substrate of NanoLuc, was used according to the manufacturer protocol in in vitro screens. Briefly, cells were seeded into 96-well solid white plates (Corning) 24 h prior the luciferase assay. Full media without phenol red was used. Prior to measurement, all plates were allowed to reach ambient temperature and the NanoGlo reagent with Furimazine (Promega) was added in 1:1 ratio to the cells. Cells were briefly shaken, incubated for 10 min, and the luminescence was measured on an EnVision plate reader (Perkin Elmer).
For luminescence measurements in vivo, 50x diluted Furimazine was used, diluted in 5% ethanol instead of the NanoGlo lysis reagent. Zebrafish embryos and larvae were measured at 1 day post injection (dpi) and 6 dpi. They were thoroughly washed in E3 medium, anesthetized by 1x Tricaine (ethyl 3-aminobenzoate methanesulfonate/MS-222, 0.16 mg/ml) and distributed into wells of a 96-well solid white plate (Corning) in approximately 50 µL of E3/1x Tricaine. Furimazine was added in a ratio 1:2 to the embryos, the plates were briefly shaken and incubated for 10 min at room temperature. Luminescence was measured on EnVision.
For luminescence measurements in vivo, 50x diluted Furimazine was used, diluted in 5% ethanol instead of the NanoGlo lysis reagent. Zebrafish embryos and larvae were measured at 1 day post injection (dpi) and 6 dpi. They were thoroughly washed in E3 medium, anesthetized by 1x Tricaine (ethyl 3-aminobenzoate methanesulfonate/MS-222, 0.16 mg/ml) and distributed into wells of a 96-well solid white plate (Corning) in approximately 50 µL of E3/1x Tricaine. Furimazine was added in a ratio 1:2 to the embryos, the plates were briefly shaken and incubated for 10 min at room temperature. Luminescence was measured on EnVision.