Fluorescence Microplate Assays

Guidelines for optimizing for fluorescence-based microplate assays

1. Select optimal fluorescence excitation and emission filters or wavelengths. Wavelength data for excitation and emission are provided in the Product Manual and/or Quick Review Cards. Poor spectral matches between detection reagents and instruments can result in large losses of signal.
2. Adjust instrument sensitivity settings to maximize fluorescence signal. To achieve maximum linear dynamic range, choose the highest gain or sensitivity setting that is suitable for both the lowest and highest anticipated assay values.
3. Use uniform assay volumes. Small differences in assay volume can cause large differences in signal. To obtain reliable measurements, always use at least the minimum volume recommended by the instrument manufacturer.
4. Mix samples thoroughly. Poor mixing can result in aggregation, precipitation, variations in reaction rates, or well-to-well concentration differences of the analyte or detection reagent.
5. Avoid bubbles. Bubbles in assay solutions cause light scattering and erroneous signals. Briefly centrifuge the microplate, degas solutions prior to dispensing (but not for live cells assay), or pop large bubbles with a pipette tip.
6. Prepare replicate samples. Analyzing replicates increases the precision of the measurements.
7. Avoid “edge effects.” A solution of a spectrally appropriate fluorophore can be used to determine the consistency of the fluorescence signal obtained across all wells of the microplate. If signal differences are observed in wells along the microplate edges, apply an appropriate calibration factor or do not use those wells.
8. Segregate bright samples. In transparent microplates, intensely fluorescent samples can affect signals observed in nearby wells (well-to-well crosstalk). Leave empty or blank wells next to intensely fluorescent samples, or load samples with similar fluorescence intensities in neighboring wells. Use white- or black-walled microplated instead of clear plates.
   
9. Avoid photobleaching samples. Do not rerun samples unless absolutely necessary. Do not rerun standards, as significant photobleaching can occur with some reagents during fluorescence measurements.
10. Use sample concentrations within the range of the assay. For unknown samples, try several dilutions.