FIGURE 2

FIGURE 2

Workflow of in vivo small-molecule using bioluminescence screening platform. The workflow of in vivo small-molecule screening started with cancer cell transplantation into 2 days post-fertilization (dpf) zebrafish embryos. The transplanted embryos were washed and kept in incubator overnight. At day 1 post-injection (dpi) the embryos were sorted according to fluorescence under an Olympus macroscope and were divided into wells of a 96-well solid white plate. Uninjected embryos in E3 water and E3 water without embryos were used as negative controls to determine the background level of luminescence. Luciferase assay was carried out using the Furimazine substrate by adding it into all wells equally and the luminescence was measured after 10 min of incubation on the EnVision plate reader. After the measurement, embryos were recovered, washed and randomly divided into groups of 6 animals into 24-well polystyrene plates, where they were treated by inhibitors from a library of kinase inhibitors at the final concentration of 10 µM and DMSO was used as negative control. We used dabrafenib and imatinib as positive controls. At the end of the experiment, at 6 dpi, the luminescence of whole embryos was measured again to determine the cell growth or its inhibition. The lower the final luminescence, compared to positive controls treated with DMSO, the stronger is the inhibitory effect. Finally, the compounds can be analyzed in vitro to determine dose response curves and to compare the in vitro vs. in vivo effectivity. Figure created in bioRENDER.