Homogeneous fluorescence readouts for miniaturized high-throughput screening: theory and practice

Pope, Andrew J.; Haupts, Ulrich; Moore, Keith J. M.

doi:10.1016/s1359-6446(99)01340-9

Cited by 191 publications

(101 citation statements)

References 6 publications

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“…17 These properties allow greater assay accuracy and sensitivity, which may enable signal detection even in the cellular environment. Furthermore, the FAM-tetramethylrhodamine (TAMRA) pair has considerably longer Fö rster radius (distance at which 50% of internal quenching occurs), allowing longer peptides (14 amino acids vs 9-10 for Dabcyl-Edans or Mca-Dnp pairs) between donor and acceptor 17 and leading to improved specificity. The larger size of FAM and TAMRA may also constrain substrate cleavage by unrelated enzymes.…”

mentioning

confidence: 99%

Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells

Alvarez-Iglesias

Wayne

O’Dea

et al. 2005

Laboratory Investigation

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Tumor necrosis factor-a (TNF) converting enzyme (TACE) is responsible for shedding of various membrane proteins including proinflammatory cytokine TNF. In vivo regulation of TACE is poorly understood mainly due to lack of reliable methodology to measure TACE activity in cell-based assays. Here we report a novel enzyme assay that enables continuous real-time measurement of TACE activity on the surface of live cells. Cells were incubated with a new fluorescent resonance energy transfer peptide consisting of a TACE-sensitive TNF sequence and fluorescein-tetramethylrhodamine (FAM-TAMRA), and enzyme activity was monitored by the rate of increase in fluorescent signal due to peptide cleavage. Validation studies using resting as well as stimulated monocytic cells indicated that the assay was sensitive, reproducible and quantitative. Pharmacological studies with various inhibitors indicated that the observed enzyme activity could largely be ascribed to TACE. Thus, the FAM-TAMRA peptide provides a powerful tool for measurement of constitutive and inducible cellular TACE activity. The principles developed may be applied to analyses of enzyme activity of various sheddases on live cells. Keywords: ADAM; ectodomain shedding; enzyme activity; FRET; protease; TACE; TNF Ectodomain shedding of membrane proteins is recognized as a potent mechanism for downregulation of their cell-associated activity while at the same time enabling their function as soluble mediators. 1,2 Tumor necrosis factor-a (TNF) converting enzyme (TACE), a member of the ADAM (a disintegrin and metalloprotease) family of proteases (ADAM-17), is the first discovered mammalian sheddase, responsible for cleavage of a variety of membrane proteins, including the proinflammatory cytokine TNF, transforming growth factor-a, p75 TNF receptor and L-selectin. 3-8 As a TNF sheddase, TACE regulates the in vivo cleavage of membranebound proTNF to release soluble TNF, 3,4 which has been shown to play a crucial role in acute and chronic inflammation. Thus, analysis of TACE activity should provide important insights into the pathophysiology of inflammatory diseases and potentially help the future development of TACEtargeted therapies. However, the mechanism of in vivo regulation of TACE activity is not well understood, 7-9 mainly due to lack of accurate methods for measuring TACE activity on viable cells. Fluorescence resonance energy transfer (FRET) peptides provide useful tools for kinetic studies of metalloproteases in solution. 10,11 These substrates consist of a donor fluorophore and a light-absorbing acceptor, attached to the terminal residues of a peptide susceptible to cleavage by a protease. Once the substrate is cleaved, increased fluorescence is observed due to loss of internal quenching, allowing quantitative measurement of real-time enzyme activity. The common donor/acceptor pairs are 4-(dimethylaminoazo) benzene-4-carboxyl (Dabcyl) and 5-(2-aminoethylamino)-1-naphthalenesulfonic (Edans), and (7-methoxycoumarin-4-yl) acetyl (Mca) and 2,4-dinitrophenyl (Dnp) or 3-...

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mentioning

confidence: 99%

Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells

Alvarez-Iglesias

Wayne

O’Dea

et al. 2005

Laboratory Investigation

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“…5 Life Imaging Center, Institute of Biology I, Developmental Biology, Freiburg, Germany. 6 To whom correspondence should be addressed. E-mail: ute.resch@ bam.de non-or minimally invasive character, and the remote accessibility of signals employing conventional optics and fiber optics.…”

Section: Federal Institute For Materials Research and Testing Workinmentioning

confidence: 99%

“…Commonly recognized advantages of fluorescence are its high sensitivity that allows even the detection of single molecules, its intrinsic selectivity concerning experimental parameters such as excitation and emission wavelength as well as fluorescence lifetime and (de)polarization [6], its ease of use, 1 …”

Section: Introductionmentioning

confidence: 99%

How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards

et al. 2005

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The scope of this paper is to illustrate the need for an improved quality assurance in fluorometry. For this purpose, instrumental sources of error and their influences on the reliability and comparability of fluorescence data are highlighted for frequently used photoluminescence techniques ranging from conventional macro-and microfluorometry over fluorescence microscopy and flow cytometry to microarray technology as well as in vivo fluorescence imaging. Particularly, the need for and requirements on fluorescence standards for the characterization and performance validation of fluorescence instruments, to enhance the comparability of fluorescence data, and to enable quantitative fluorescence analysis are discussed. Special emphasis is dedicated to spectral fluorescence standards and fluorescence intensity standards.

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“…12 An advantage of fluorescence spectroscopy is that different assays can be designed based on different aspects of the fluorescence output (lifetime, intensity, anisotropy and energy transfer). 11,13 Additionally, laser fiber optics and detection technologies are well established. Therefore, fluorescence techniques are envisioned as the most important future detection method for miniaturized ultra-high-throughput screening.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, fluorescence techniques are envisioned as the most important future detection method for miniaturized ultra-high-throughput screening. 13 Chemical sensing using fluorescence to signal a molecular recognition event was first demonstrated during the early 1980s when Tsien et al reported the synthesis of the first fluorescent calcium indicators. 14,15 They are based on calcium ion chelate receptors, covalently linked to simple aromatics rings or other dyes as chromophores.…”

Section: Introductionmentioning

confidence: 99%

Design of Fluorescent Materials for Chemical Sensing

2007

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There is an enormous demand for chemical sensors for many areas and disciplines. High sensitivity and ease of operation are two main issues for sensor development. Fluorescence techniques can easily fulfill these requirements and therefore fluorescent-based sensors appear as one of the most promising candidates for chemical sensing. However, the development of sensors is not trivial; material science, molecular recognition and device implementation are some of the aspects that play a role in the design of sensors. The development of fluorescent sensing materials is increasingly captivating the attention of the scientists because its implementation as a truly sensory system is straightforward. This critical review shows the use of polymers, sol-gels, mesoporous materials, surfactant aggregates, quantum dots, and glass or gold surfaces, combined with different chemical approaches for the development of fluorescent sensing materials. Representative examples have been selected and they are commented here.

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Homogeneous fluorescence readouts for miniaturized high-throughput screening: theory and practice

Cited by 191 publications

References 6 publications

Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells

Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells

How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards

Design of Fluorescent Materials for Chemical Sensing

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