Assessing Neuronal Excitability on a Fluorometric Imaging Plate Reader (FLIPR) Following a Defined Electrostimulation Paradigm

2019 ◽  
pp. 219-226
Author(s):  
Valeria Kizner ◽  
Sandra Fischer ◽  
Benjamin Jähnke ◽  
Maximilian Naujock
Keyword(s):  
Neuronal Excitability ◽  
Imaging Plate ◽  
Plate Reader

scholarly journals Use of a Fluorescent Imaging Plate Reader-Based Calcium Assay to Assess Pharmacological Differences between the Human and Rat Vanilloid Receptor

2002 ◽  
Vol 7 (5) ◽  
pp. 466-475 ◽  
Author(s):  
David G. Witte ◽  
Steven C. Cassar ◽  
Jeffrey N. Masters ◽  
Timothy Esbenshade ◽  
Arthur A. Hancock
Keyword(s):  
Ruthenium Red ◽  
Vanilloid Receptor ◽  
Fluorescent Imaging ◽  
Imaging Plate ◽  
Hek 293 ◽  
Electrophysiological Properties ◽  
Competitive Antagonism ◽  
Rapid Responses ◽  
Plate Reader ◽  
Highly Correlated

The cloned vanilloid receptor 1 (VR1) is a ligand-gated calcium channel that is believed to be the capsaicin-activated vanilloid receptor found in native tissues, based on similarities regarding molecular mass, tissue distribution, and electrophysiological properties. Using a Fluorescent Imaging Plate Reader (FLIPR), along with Fluo-3 to signal intracellular calcium levels ([Ca++]i), rat VR1 (rVR1) and a human orthologue (hVR1) were pharmacologically characterized with various VR1 ligands. HEK-293 cells, stably expressing rVR1 or hVR1, exhibited dose-dependent increases in [Ca++]i when challenged with capsaicin (EC50s ≅ 10 nM). Responses to capsaicin were blocked by the VR1 antagonist capsazepine and were dependent on VR1 expression. Potencies for 10 structurally diverse VR1 agonists revealed rVR1 potencies highly correlated to that of hVR1 ( R2 = 0.973). However, a subset of agonists (tinyatoxin, gingerol, and zingerone) was approximately 10-fold more potent for rVR1 compared to hVR1. Schild analysis for blockade of capsaicin-induced responses by capsazepine was consistent with competitive antagonism, whereas ruthenium red displayed noncompetitive antagonism. Compared to rVR1, hVR1 was more sensitive to blockade by both antagonists. For both rVR1 and hVR1, time-response waveforms elicited by resiniferatoxin increased more gradually compared to other agonists. Tinyatoxin also displayed slow responses with hVR1 but showed rapid responses with rVR1. Thus, FLIPR technology can be used to readily reveal differences between rVR1 and hVR1 pharmacology with respect to potencies, efficacies, and kinetics for several VR1 ligands.

Measuring Ca2+ Changes in Multiwell Format Using the Fluorometric Imaging Plate Reader

2005 ◽  
pp. 125-132
Author(s):  
Ian C. B. Marshall ◽  
Davina E. Owen ◽  
Shaun McNulty
Keyword(s):  
Imaging Plate ◽  
Plate Reader

A High-Throughput Screening Campaign for Detection of Ca2+–Activated K+ Channel Activators and Inhibitors Using a Fluorometric Imaging Plate Reader–Based Tl+-Influx Assay

2013 ◽  
Vol 11 (3) ◽  
pp. 163-172 ◽  
Author(s):  
Susanne Jørgensen ◽  
Tino Dyhring ◽  
David T. Brown ◽  
Dorte Strøbæk ◽  
Palle Christophersen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
K Channel ◽  
Imaging Plate ◽  
Plate Reader

Evaluation and improvements of the Rigaku imaging plate reader (R-Axis IV++) for the use in synchrotron X-ray solution scattering

2003 ◽  
Vol 36 (3) ◽  
pp. 535-539 ◽  
Author(s):  
Tetsuro Fujisawa ◽  
Yukihiro Nishikawa ◽  
Hideto Yamazaki ◽  
Yoji Inoko
Keyword(s):  
Imaging Plate ◽  
X Ray ◽  
Plate Reader

A Thallium Transport FLIPR-Based Assay for the Identification of KCC2-Positive Modulators

2010 ◽  
Vol 15 (2) ◽  
pp. 177-184 ◽  
Author(s):  
Di Zhang ◽  
Sujatha M. Gopalakrishnan ◽  
Gail Freiberg ◽  
Carol S. Surowy
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Inhibitory Neurons ◽  
Imaging Plate ◽  
Neuronal Excitation ◽  
Inhibitory Mechanisms ◽  
Pharmacological Studies ◽  
Plate Reader ◽  
Transport Assay

KCC2, potassium chloride cotransporter 2, is expressed exclusively in the CNS (on inhibitory neurons) and plays a major role in maintaining appropriately low intracellular chloride levels that ensure inhibitory actions of GABAA and glycine receptors. As such, it plays a pivotal role in inhibitory mechanisms that control neuronal excitation in the CNS. KCC2 downregulation has been implicated in various excitatory disorders, such as epilepsy and neuropathic pain. Positive modulators of KCC2 expression or activity may thus provide effective therapy for these disorders. However, the identification of such agents is hindered by the lack of a high-throughput screening method. Here the authors report the development of a fluorescence-based thallium (Tl+) transport assay using a Fluorometric Imaging Plate Reader (FLIPR), in which KCC2 activity is assessed by measuring the initial rate of KCC2-mediated Tl+ transport/influx. The authors demonstrate Tl+/Cl− cotransport by KCC2, which exhibits a high apparent affinity for Tl+ and dependency on the presence of the Cl− ion. Pharmacological studies revealed anticipated effects and potencies of known KCC-positive (NEM, staurosporine) and KCC-negative (DIOA, furosemide) modulators. The authors demonstrate that the assay is robust and reproducible and can be employed in high-throughput screening for positive modulators of KCC2 as potential therapeutic agents.

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

1999 ◽  
Vol 4 (5) ◽  
pp. 249-258 ◽  
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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