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Related Concept Videos

Immunofluorescence Microscopy01:12

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A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.
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Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland
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Avoiding Fluorescence Assay Interference-The Case for Diaphorase.

Matthew D Hall1, Anton Simeonov1, Mindy I Davis1

  • 1NCATS Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland.

Assay and Drug Development Technologies
|April 15, 2016
PubMed
Summary
This summary is machine-generated.

High-throughput screening (HTS) assays can suffer interference from library compounds, especially those using UV excitation. This study highlights the underutilized diaphorase/resazurin system as a robust solution to avoid fluorescence interference in NADH/NADPH-dependent assays.

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Area of Science:

  • Biochemistry
  • Assay Development
  • High-Throughput Screening

Background:

  • Fluorescence-based assays are crucial for high-throughput screening (HTS).
  • Assay interference from screening compounds is a significant challenge, particularly for UV-excited assays detecting oxidoreductase cofactors like NADH and NADPH.
  • Direct detection of NADH/NADPH can be prone to compound interference.

Purpose of the Study:

  • To discuss challenges associated with fluorescence interference in HTS assays.
  • To highlight the diaphorase/resazurin system as a potential solution for NADH/NADPH-dependent assays.
  • To advocate for increased utilization of the diaphorase/resazurin system in assay development.

Main Methods:

  • Literature review of the diaphorase/resazurin assay system.
  • Analysis of fluorescence interference issues in HTS.
  • Discussion of coupling strategies for NADH/NADPH-dependent enzymes.

Main Results:

  • Compound interference is a recognized problem in fluorescence HTS.
  • The diaphorase/resazurin system offers a sensitive and robust method for coupling to NADH/NADPH-dependent enzymes.
  • Existing literature suggests the diaphorase/resazurin system is underutilized.

Conclusions:

  • The diaphorase/resazurin system is a valuable, yet underutilized, tool for developing robust fluorescence assays.
  • Employing this system can mitigate fluorescence interference issues common in HTS.
  • Further adoption of this assay system is encouraged to improve HTS reliability.