BlueScreen HC is based on the fast, accurate GreenScreen HC genotoxicity assay from Gentronix Ltd. BlueScreen HC uses the same human-derived, p53-competent TK6 cells to host a patented reporter system that exploits the proper regulation of the GADD45a gene. However, in place of GFP, BlueScreen HC utilizes a luciferase from the marine copepod Gaussia princeps to generate a luminescent output. The luminescent assay delivers both high specificity and high sensitivity and detects all common mechanistic classes of genotoxin, just like GreenScreen HC. The 96-well microplate format is rapid to set up and results are generated after 48 hours’ incubation. A single microplate is sufficient for the simultaneous testing of 4 compounds and the protocol is readily automated using standard laboratory equipment. The superior signal-to-noise ratio makes it more suitable for adaptation to even higher throughput screening in 384-well format. The luminescent output also circumnavigates issues of test compound autofluorescence.
Key Principles:
GADD45a plays an important role in mediating the adaptive response to genotoxic stress. The patented system incorporates complex regulatory elements to enable a faithful GADD45a response. The assay generates positive results for direct-acting mutagens, clastogens, as well as aneugens, and topoisomerase and polymerase inhibitors. Importantly, correct negative results are produced for non-carcinogens, many of which give misleading positive results in other in vitro genotoxicity tests. A metabolic activation protocol using rodent S9 liver extract extends the range of compounds detected to include genotoxic metabolites.
Assay Protocol:
The BlueScreen HC protocol permits 4 compounds to be tested across 8 dilutions (2-fold serial), together with untreated and positive controls, in one 96-well microplate. After arraying test compounds and dilutions, growing cells are added to each well. For studies without S9 metabolic activation data are collected after incubation (48h) using a multimode microplate reader. For studies incorporating S9 metabolic activation, cells are washed after a 3h incubation with test compound and S9. Measurements are made by microplate reader after a further 45h incubation period. Simple software gives automated decisions and a clear graphical output, including a complimentary indication of cytotoxicity (RCD).