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

a laser scanning confocal microscope or similar instrument. A weak signal thus precludes use in a microplate spectrofluorometer, which would allow increased throughput. The SPECTRAmax GEMINI-XS is a dual-monochromator microplate fluorometer system incorporating highly sensitive optics4 that permit experimental determinations at low-level detection limits. This application note describes experimental protocols that enable the detection of relatively low numbers of cells expressing Green Fluorescent Protein (GFP) in a 96-well microplate using the SPECTRAmax GEMINI-XS spectrofluorometer.


MATERIALS AND METHODS
Human embryonic kidney 293 cells (HEK-293) were transiently transfected with a wild-type GFP expression plasmid (pS65T, Clontech, discontinued product; cytomegalovirus immediate early promoter drives expression of GFP) using Lipofectamine as per manufacturer's instructions (Gibco/Invitrogen, Cat. No. 18324-012). Cells were grown to confluence in 162 cm2 flasks under standard cell-culture conditions. Fluorescence microscopy revealed an expression level in excess of 75 %.

The GFP-expressing cells were removed with trypsin, and the cells were washed with medium (MEM Alpha, Gibco-RBL Cat. No. 22571-020). The appropriate number of cells was seeded in alternate columns of a 96-well microplate (Costar, black walled, clear bottomed Cat. No. 3603) and diluted with mock-transfected cells to maintain a constant cell number of 200,000 cells per well. This resulted in a final number of 6,250 (column 12) to 200,000 (column 2) GFP expressing cells per well in a volume of 100 l. These cells were seeded in alternate columns 2, 4, 6, 8, 10, and 12. In contrast, columns 1, 3, 5, 7, 9 and 11 were seeded with 200,000 mock-transfected cells per well. Then the plate was returned to the incubator for 48 hours.

Initially, excitation and emission scans were run to select the optimum excitation wa
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