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

velength and emission wavelength/cutoff filter combination for the maximum signal/background ratio5. The parameters selected for all subsequent experiments were: GFP; λΕX 472 nm, λΕM 512 nm with a 495 nm emission cutoff filter.


RESULTS
Results show a linear relationship between fluorescence intensity and cell number (Fig.1) A maximal fluorescence intensity of 177 3 RFU was observed at the highest cell concentration (Table 1). SPECTRAmax GEMINI-XS spectrofluorometer was able to detect a specific GFP signal at all cell numbers as determined by One Way Analysis of Variance (ANOVA) (Table 1). This analysis indicated that, after 48 hours incubation, there was a significant difference (P < 0.01) between the GFP-transfected and mock-transfected cells at an initial seeding density of 6,250 GFP- expressing cells per well (Table 1). Since approximately 75 % of the cells actually express GFP, this translates to a detection limit of roughly 4,690 GFP-expressing cells/well.



A set of experiments was designed to compare two types of microplates for their ability to detect a 1 M fluorescein solution compared to suspension of GFP-expressing cells (Table 2). Interestingly, the fluorescein signal increased from 11190 RFU in Greiner plates to 32787 RFU in ABgene Thermo-Fast 96 skirted black PCR plates (Cat. No. AB-0800, specify black when ordering). More importantly, the signal detected from GFP-expressing cells increased by almost 3fold when black ABgene PCR plates were used (Table 2). A possible explanation for the greater signal detectable using the black PCR plates is that the base of each well of the PCR plate is higher and the wells are narrower than the standard microplate. This feature brings the sample closer to the optics and for a constan
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