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Measurement of Green Fluorescent Protein in the SpectraMax Gemini XS Spectrofluorometer (MaxLine Application Note #44)

t volume, effectively increases the path-length.


DISCUSSION
GFP and its variants are convenient markers for use in the fluorescent detection of gene expression or protein localization in living cells because it eliminates the need to incubate with secondary reagents such as dyes or antibodies. Additionally, vectors encoding these fluorescent proteins are also commonly used as simple transfection markers. Furthermore, GFP fluorescence is stable, species-independent, and does not require any substrates or cofactors. With the burgeoning use of GFP in life sciences and drug discovery research, and ever increasing demands for higher assay throughputs, traditional detection technology such as fluorescence-activated cell sorting (FACS) and fluorescence microscopy can no longer satisfy demands. Transfer of an assay to 96-well microplate technology increases throughput significantly.

This report demonstrates that the SPECTRAmax GEMINI-XS microplate spectrofluorometer, with its dual scanning monochromator and patented optical sensitivity, can be used to detect a relatively low number of cells expressing GFP. The proprietary optical design with the use of elliptical mirrors instead of lenses enhances sensitivity and data reproducibility. Even at an initial seeding density of 6,250 GFP-expressing cells per well, the Gemini-XS is able to reliably detect the 1.2-fold increase in signal over mock transfected cells. Because the SPECTRAmax GEMINI-XS is able to monitor up to four different wavelengths during an experiment, it may be used for multiple color-labeling experiments such as those using the range of Clontech Living Colors Fluorescent Proteins.

In practical terms, possible uses of the SPECTRAmax GEMINI-XS with fluorescent proteins include discrimination of cellular apoptosis or necrosis (apoptosis induced decrease in fluorescence6), determination of bacterial survival
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