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Fluorescence Quenching in Cell-Based Assays Can Be Dye Specific: A Case Study using Both Fura-2 AM and Fluo-4 AM Calcium Reporter Dyes Using the FDSS6000

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Fluorescence Quenching in Cell-Based Assays Can Be Dye Specific: A Case Study using Both Fura-2 AM and Fluo-4 AM Calcium Reporter Dyes Using the FDSS6000

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Fluorescence quenching refers to any process which decreases the fluorescence intensity of a given substance. Quenchers may not affect all fluorophores, allowing for selective quenching. There are two types of quenching: Static and Dynamic. Static quenching occurs when the fluorophore binds to the quencher; upon light absorption (excitation) fluorophore returns to the ground state with no photon emission (fluorescence). Dynamic quenching describes reducing the fluorescence lifetime of the fluorophore, that is the fluorophore emits photons for less time (decrease in 'fluorescence lifetime') than in the absence of a quencher. Diverse compound libraries used in High Throughput Screening (HTS) for antagonists may contain fluorophore quenchers, generating results apparently indistinguishable from real antagonist hits (false positive). Further, in agonist screening campaigns quenchers may mask true positive agonist hits (false negatives), an all important consideration in assay design.

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