Fluorescence Plate Reader for Quantum Dot-Protein Bioconjugation Analysis

Intramolecular CFP-YFP fluorescence resonance energy transfer (FRET) sensors expressed in cells are powerful research tools but have seen relatively little use in screening. We exploited the discovery that the expression of a CFP-YFP FRET diacylglycerol sensor (DAGR) increases over time when cells are incubated at room temperature to assess requirements for robust measurements using a Molecular Devices Spectramax i3x fluorescence plate reader. Expression levels resulting in YFP fluorescence >10-fold higher than untransfected cells and phorbol ester-stimulated FRET ratio changes of 60% or more were required to consistently give robust Z′ > 0.5. As a means of confirming that these conditions are suitable for screening, we developed a novel multiple-read protocol to assay the NCI’s Mechanistic Set III for agonists and antagonists of C1 domain activation. Sixteen compounds prevented C1 domain translocation. However, none blocked phorbol ester-stimulated protein kinase C (PKC) activity assessed using a phospho-specific antibody—six actually stimulated PKC activity. Cytometry, which produces higher Z′ for a given FRET ratio change, might have been a better approach for discovering antagonists, as it would have allowed lower phorbol ester concentrations to be used. We conclude that CFP-YFP FRET measured in a Spectramax i3x plate reader can be used for screening under the conditions we defined. Our strategy of varying expression level and FRET ratio could be useful to others for determining conditions needed for robust cell-based intramolecular CFP-YFP FRET measurements on their instrumentation.

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A novel cycling assay for cellular cADP-ribose with nanomolar sensitivity

Biochemical Journal ◽

10.1042/bj3610379 ◽

2002 ◽

Vol 361 (2) ◽

pp. 379-384 ◽

Cited By ~ 94

Author(s):

Richard GRAEFF ◽

Hon Cheung LEE

Keyword(s):

High Sensitivity ◽

Physiological Conditions ◽

Adp Ribosyl Cyclase ◽

High Throughput Method ◽

Plate Reader ◽

Coupled Reactions ◽

High Concentrations ◽

Nanomolar Range ◽

Fluorescence Plate Reader

cADP-ribose (cADPR) is a novel cyclic nucleotide derived from NAD+ that has now been established as a general Ca2+ messenger in a wide variety of cells. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, its measurement has been technically difficult and requires specialized reagents. In this study a widely applicable high-sensitivity assay for cADPR is described. ADP-ribosyl cyclase normally catalyses the synthesis of cADPR from NAD+, but the reaction can be reversed in the presence of high concentrations of nicotinamide, producing NAD+ from cADPR stoichiometrically. The resultant NAD+ can then be coupled to a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD+ cycles through these coupled reactions, a molecule of highly fluorescent resorufin is generated. The reaction can be conducted for hours, resulting in more than a thousand-fold amplification of cADPR. Concentrations of cADPR in the nanomolar range can be measured routinely. The unique ability of ADP-ribosyl cyclase to catalyse the reverse reaction provides the required specificity. Using this assay, it is demonstrated that cADPR is present in all tissues tested and that the levels measured are directly comparable with those obtained using a radioimmunoassay. All the necessary reagents are widely available and the assay can be performed using a multiwell fluorescence plate reader, providing a high-throughput method for monitoring cADPR levels. This assay should be valuable in elucidating the messenger role of cADPR in cells.

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Using 96-well tissue culture polystyrene plates and a fluorescence plate reader as tools to study the survival and inactivation of viruses on surfaces

Cytotechnology ◽

10.1007/s10616-011-9355-8 ◽

2011 ◽

Vol 63 (4) ◽

pp. 385-397 ◽

Cited By ~ 15

Author(s):

Phuc H. Pham ◽

Junwon Jung ◽

Niels C. Bols

Keyword(s):

Tissue Culture ◽

Tissue Culture Polystyrene ◽

Plate Reader ◽

Fluorescence Plate Reader

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An optimized high-throughput fluorescence plate reader-based RSV neutralization assay

Journal of Virological Methods ◽

10.1016/j.jviromet.2018.07.004 ◽

2018 ◽

Vol 260 ◽

pp. 34-40 ◽

Cited By ~ 1

Author(s):

Yong-Peng Sun ◽

Wei Zhang ◽

Qin-Jian Zhao ◽

Jian-Li Cao ◽

Lu-Jing Zhang ◽

...

Keyword(s):

High Throughput ◽

Neutralization Assay ◽

Plate Reader ◽

Fluorescence Plate Reader

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A novel cycling assay for nicotinic acid–adenine dinucleotide phosphate with nanomolar sensitivity

Biochemical Journal ◽

10.1042/bj20020644 ◽

2002 ◽

Vol 367 (1) ◽

pp. 163-168 ◽

Cited By ~ 36

Author(s):

Richard GRAEFF ◽

Hon Cheung LEE

Keyword(s):

Nicotinic Acid ◽

Hydrolytic Enzymes ◽

High Sensitivity ◽

Adp Ribosyl Cyclase ◽

High Throughput Method ◽

Plate Reader ◽

Coupled Reactions ◽

High Concentrations ◽

Micromolar Range ◽

Fluorescence Plate Reader

Nicotinic acid—adenine dinucleotide phosphate (NAADP) is a novel nucleotide derived from NADP that has now been shown to be active in releasing Ca2+ from intracellular stores in a wide variety of cells ranging from plant to human. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, no assay has been reported for NAADP to date. In the present study, a widely applicable assay for NAADP with high sensitivity is described. NAADP was first dephosphorylated to nicotinic acid—adenine dinucleotide by treatment with alkaline phosphatase. The conversion was shown to be stoichiometric. NMN-adenylyltransferase was then used to convert nicotinic acid—adenine dinucleotide into NAD in the presence of high concentrations of NMN. The resultant NAD was amplified by a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD cycled through these coupled reactions, a molecule of highly fluorescent resorufin was generated. The reaction could be performed for hours, resulting in more than a 1000-fold amplification. Concentrations of NAADP over the 10—20nM range could be routinely measured. This novel cycling assay was combined with an enzymic treatment to provide the necessary specificity for the assay. NAADP was found to be resistant to NADase and apyrase. Pretreatment of samples with a combination of the hydrolytic enzymes completely eliminated the interference from common nucleotides. The versatility of the cycling assay can also be extended to measure nicotinic acid, which is a substrate in the synthesis of NAADP catalysed by ADP-ribosyl cyclase, over the micromolar range. All the necessary reagents for the cycling assay are widely available and it can be performed using a multi-well fluorescence plate reader, providing a high-throughput method. This is the first assay reported for NAADP and nicotinic acid, which should be valuable in elucidating the messenger functions of NAADP.

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