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Fluorescence Microplate AssaysSubstrates, buffers, and calibration standards for kinetic or endpoint assays across various biological applications |
Combining the sensitivity of fluorescence-based assays and convenience of microplate format allows researchers to achieve rapid and quantitative readout using high-throughput analysis. Within a microplate well, the fluorescent signal can be generated with intact cells, cell lysates, or purified enzyme preparations, and subsequently quantified by measuring the fluorescence intensity emitted from the well without the need for cellular imaging.
A variety of fluorescence microplate assays is available, from cell viability and proliferation assays to enzyme activity and protein quantification assays. These tools offer accurate and reliable measurements for kinetic and endpoint protocols across a wide range of biological applications.
Fluorescence microplate applications
Measure the activity of various analytes including cholesterol, phosphates and pyrophosphate, phosphatase, and phospholipase
Use a variety of assays to evaluate viability such as reducing or redox potential, release of enzymatic proteins or membrane integrity
Assays for the measurement of protease and other enzymes such as collagenases, elastases, lysozymes, reverse transcriptase, and RNA-dependent RNA polymerase
Assays and substrates for the sensitive detection of β-galactosidase or glucosidases
Track intracellular calcium and magnesium, or measure intracellular pH with a range of wavelength options in live cells
Amplex Red substrate is used in various metabolic, neurobiology, and inflammation assays to detect a variety of analytes such as glucose, galactose, cholesterol, glutamic acid, xanthine (or hypoxanthine), uric acid, choline and acetylcholine, as well as hydrogen peroxide
Assays to quantify dsDNA, ssDNA, and RNA for downstream applications such as next-generation sequencing, transfection, real-time PCR, microarray experiments, RT-PCR, differential display PCR, northern blot, S1 nuclease assays, RNase protection assays, and cDNA library preparations
Quantitation kits that are specific for components within the sample; for example, CBQCA is compatible with lipoproteins or lipid protein mixtures, while NanoOrange is compatible with reducing agents, and the Quant-iT assay is compatible with salts and solvents but not detergents
Detection of a variety of reactive oxygen species (ROS) such as generalized oxidative stress, nitric oxide (NO), reduced glutathione (GSH), and myeloperoxidase (MPO)
Expertly detect fluorescence with Thermo Scientific plate readers
High-sensitivity fluorescence detection for 6-1,536 samples can be quickly performed on the Varioskan ALF or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents to enable optimal detection. Take advantage of automatic dynamic range selection to get optimal gain settings for each individual well and automation capabilities for even higher throughput.
Guidelines for optimizing for fluorescence-based microplate assays
- 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.
- 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.
- 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.
- 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.
- 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.
- Prepare replicate samples. Analyzing replicates increases the precision of the measurements.
- 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.
- 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.
- 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.
- Use sample concentrations within the range of the assay. For unknown samples, try several dilutions.
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