Key words: fluorescence • LEADseeker • Cy • Alexa Fluor • TAMRA • Brij
Flexible and scalable uHTS programs demand that organizations be able to select the most appropriate or cost-effective fluorescent reagents for the task. This application note describes experiments to assess the performance of a variety of dyes with LEADseeker™ Multimodality Imaging System. Data was collected in the system's fluorescence intensity and fluorescence polarization modes. Lowest limits of detection (LLD) were determined for the dyes using either the standard filter sets supplied with the instrument, or a collection of filter configurations defined specifically for each dye type. Results demonstrate that a variety of dyes are fully compatible with the LEADseeker Multimodality Imaging System, rendering it a versatile detection system capable of supporting a broad range of uHTS applications.
LEADseeker Multimodality Imaging System 18-1140-71
Cy™3B; Cy5 PA63101; PA15101
Alexa FluorTM 532 optical configuration
(for FP modality) consists of: Fluorescence
excitation filter 500 nm, bp 40 nm Fluorescence
perpendicular dichroic Fluorescence emission
filter 568 nm, bp 50 nm
Alexa Fluor 532 Fluorescence Epi-mirror dichroic 11-0013-73
Fluorescence Epi-mirror dichroic for
BODIPYTM-TMR (in FP modality)
Other materials required
Alexa Fluor 488, 532, 546, 555, and 647 (Molecular Probes)
BODIPY-TMR and BODIPY-650/665 (Molecular Probes)
Fluorescein and TAMRA™ (Molecular Probes)
All of the fluorescent dye reagents were dissolved from powder form into DMSO at approximately 10 mg/ml. Subsequently, each dye was diluted in buffer (PBS containing 0.01% [v/v] Brij™-35) to produce solutions for determining concentration. In all cases, we determined concentration by measuring absorbance (at the absorption maxima for the dye) against a buffer-only reference and by applying the Beer Lambert equation:
i.e. c = O.D. / εl where:
c = concentration
O.D. = absorption value determined
ε = extinction coefficient of the relevant fluorescent dye
Following determination of concentration, each dye was diluted further in buffer to a top working concentration of 6.4 nM. It was then subjected to doubling dilution in buffer to produce a low concentration of 0.00625 nM. Replicates of each dilution and buffer-only reference were added (as a total volume of 50 µl per well) to a 384-well microplate and imaged (using appropriate optical components) on LEADseeker Multimodality Imaging System in either fluorescence intensity or fluorescence polarization mode for a maximum of 10 s coincidence averaging counting.
Following imaging, we determined the mean value of the signal and standard deviation at each concentration for data obtained from each of the replicates in fluorescence intensity mode. LLD was then determined as the lowest dye concentration with an observed intensity value greater than that of the buffer + 3 SD of the buffer-only wells. Table 1 shows the dat a obtained for a variety of dyes using the standard optical configurations available with LEADseeker Multimodality Imaging System.
Performance was also assessed in fluorescence polarization mode. Here, too, we calculated the mean value of the signal and standard deviation at each concentration and then determined the LLD (where LLD was the lowest dye concentration with an observed polarization value consistent with that used as the reference value for that dye and with a standard deviation value of less than 10 mP for the replicates). Table 2 shows the data obtained for a variety of dyes using the standard optical configurations available on LEADseeker Multimodality Imaging System; data obtained using the dye-specific optical configurations is shown in Table 3.
• A variety of fluorescent dyes can be used in either fluorescence intensity or fluorescence polarization mode with LEADseeker Multimodality Imaging System.
• The ability to choose from standard filter sets or from dyespecific optical configurations enhances the flexibility of LEADseeker Multimodality Imaging System.
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