Rapid Measurements of Intracellular Calcium Using a Fluorescence Plate Reader

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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Analysis of Apparent Noncompetitive Responses to Competitive H1-Histamine Receptor Antagonists in Fluorescent Imaging Plate Reader-Based Calcium Assays

CrossRef Listing of Deleted DOIs ◽

10.1177/108705719900400506 ◽

1999 ◽

Vol 4 (5) ◽

pp. 249-258 ◽

Cited By ~ 31

Author(s):

Thomas R. Miller ◽

David G. Witte ◽

Lynne M. Ireland ◽

Chae Hee Kang ◽

Jean M. Roch ◽

...

Keyword(s):

Intracellular Calcium ◽

Histamine Receptor ◽

Fluorescent Imaging ◽

Model Systems ◽

Imaging Plate ◽

Transient Effects ◽

Response Curves ◽

Histamine Concentration ◽

Pharmacological Characterization ◽

Plate Reader

We have examined the utility of high throughput fluorescent imaging plate reader (FLIPR)-based calcium assays for pharmacological characterization of G-protein coupled receptors (GPCRs) using recombinant and native human H1-histamine receptors (H1-HR), expressed in HEK293 and HeLa S3 cells, respectively, as model systems. For stably transfected HEK293 cell lines, the potency of histamine for elevating intracellular calcium increased (pD2, 7.13 and 7.86) with increased H1-HR density (about 0.8 and 14 pmol/mg protein, respectively), though histamine binding affinities were similar. The classic H1-HR competitive antagonists diphenhydramine and chlorpheniramine appeared noncompetitive by causing depressions of the maximal histamine responses along with rightward shifts of histamine concentration-response curves, thus precluding Schild analysis. Applying the generalized Cheng-Prusoff equation to antagonist concentration-response curves for inhibition of fixed histamine concentrations yielded apparent pKb values that were consistent among recombinant and native receptors at different expression levels. These pKb values for diphenhydramine and chlorpheniramine (e.g., 7.83 and 8.77, respectively) were in good agreement with binding pKi values (e.g., 7.98 and 8.52, respectively). Apparent antagonist affinities determined from FLIPR calcium and competition binding assays were also consistent for the competitive antagonists mepyramine, tripelennamine, and promethazine. In phosphoinositide hydrolysis assays, chlorpheniramine exhibited insurmountable inhibition of histamine calcium responses, although to a lesser extent than that observed in calcium assays; pKb values were similar. These results demonstrate that competitive antagonist potencies can be attained from FLIPR-derived data by application of the generalized Cheng-Prusoff equation, despite apparent noncompetitive antagonism under these assay conditions. Apparent noncompetitive antagonist effects may in part be attributable to a lack of equilibrium of histamine and antagonists with H1-HR within the short duration of rapid transient effects of histamine on intracellular calcium.

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