Microplate based GreenScreen HC and GC assays, are designed to be compatible with standard off-the-shelf laboratory automation. They require the use of a fluorescence plate reader capable of performing both fluorescence and absorbance measurements on 96-well microplates. Following in-house evaluations, Gentronix recommends the following plate readers for use with GreenScreen HC and GC:
The GreenScreen assays each use two strains, one of which does not express GFP, to allow the software to correct for the otherwise interfering effect of autofluorescent compounds. In a small minority of cases, for analytes that are intensely autofluorescent at GFP wavelengths, a method of correction using fluorescence polarisation can be employed. This protocol exploits the high fluorescence anisotropy of the GFP molecule compared to other smaller autofluorescent species such as the test compound. For this reason it is preferable, that the plate reader used for GreenScreen is also capable of performing fluorescence polarisation readings.
Cellular fluorescence is measured using standard factory fitted filters normally used for the measurement of fluorescein, a close spectroscopic mimic of GFP - typically excitation at 485 nm and emission at 535 nm. Absorbance is measured at 600 to 620 nm.