scholarly journals Development of a Novel Cell-Based Assay System for High-Throughput Screening of Compounds Acting on Background Two-Pore Domain K+ Channels

2019 ◽  
Vol 24 (6) ◽  
pp. 641-652
Author(s):  
Keisuke Kawasaki ◽  
Yoshiaki Suzuki ◽  
Hisao Yamamura ◽  
Yuji Imaizumi
Keyword(s):  
Cell Death ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Efficiency ◽  
Equilibrium Potential ◽  
Channel Blockers ◽  
Pore Domain ◽  
Druggable Targets ◽  
Relative Cell Viability ◽  
Or Task

Two-pore domain K+ (K2P) channels are thought to be druggable targets. However, only a few agents specific for K2P channels have been identified, presumably due to the lack of an efficient screening system. To develop a new high-throughput screening (HTS) system targeting these channels, we have established a HEK293-based “test cell” expressing a mutated Na+ channel (Nav1.5) with markedly slowed inactivation, as well as a K+ channel (Kir2.1) that sets the membrane potential quite negative, close to K+ equilibrium potential. We found in this system that Kir2.1 block by 100 μM Ba2+ application consistently elicited a large depolarization like a long-lasting action potential. This maneuver resulted in cell death, presumably due to the sustained Na+ influx. When either the TWIK-related acid-sensitive K+ (TASK)-1 or TASK-3 channel was expressed in the test cells, Ba2+-induced cell death was markedly weakened. Stronger activation of TASK-1 by extracellular acidification further decreased the cell death. In contrast, the presence of K2P channel blockers enhanced cell death. IC50 values for TASK-1 and/or TASK-3 blockers acquired by measurements of relative cell viability were comparable to those obtained using patch-clamp recordings. Both blockers and openers of K2P channels can be accurately assessed with high efficiency and throughput by this novel HTS system.

Fluorescence-Based High Throughput Screening Technologies for Natural Chloride Ion Channel Blockers

2018 ◽  
Vol 31 (12) ◽  
pp. 1332-1338 ◽  
Author(s):  
Rong Xu ◽  
Yuan Xiao ◽  
Yan Liu ◽  
Bo Wang ◽  
Xing Li ◽  
...  
Keyword(s):  
Ion Channel ◽  
High Throughput ◽  
High Throughput Screening ◽  
Chloride Ion ◽  
Channel Blockers ◽  
Ion Channel Blockers

scholarly journals A Novel High-Throughput Technique for Identifying Monoclonal Antibodies capable of Death Receptor Induced Apoptosis

2009 ◽  
Vol 2 ◽  
pp. JCD.S3660
Author(s):  
Hang Fai Kwok ◽  
Julie A. Gormley ◽  
Christopher J. Scott ◽  
James A. Johnston ◽  
Shane A. Olwill
Keyword(s):  
Cell Death ◽  
High Throughput ◽  
High Throughput Screening ◽  
False Negative ◽  
Death Receptor ◽  
Annexin V ◽  
Fas Receptor ◽  
Induced Apoptosis

The study of death receptor family induced apoptosis has gained momentum in recent years with the knowledge that therapeutic antibodies targeting DR4 and DR5 (death receptor's 4 and 5) have proved efficacious in multiple clinical trials. The therapeutic rationale is based on targeting and amplifying a tumour tissues normal cell death programme (apoptosis). While advances in the targeting of DR4 and DR5 have been successful the search for an agonistic antibody to another family member, the Fas receptor, has proven more elusive. This is partly due to the differing in vitro and in vivo characteristics of individual antibodies. In order to induce Fas targeted cell death an antibody must be capable of binding to and trimerising the receptor. It has been shown that antibodies capable of performing this function in vivo, with the assistance of tumour associated cells, do not always induce apoptosis in vitro. As a result the use of current methodologies to detect functional antibodies in vitro may have dismissed potential therapeutic candidates ('false negative'). Here we report a novel high throughput screening technique which artificially cross-links antibodies bound to the Fas receptor. By combining this process with Annexin-V and Prodidium Iodide (PI) staining we can select for antibodies which have the potential to induce apoptosis in vivo.

scholarly journals Reliable high-throughput screening with by limiting yeast cell death phenomena

2004 ◽  
Vol 5 (2) ◽  
pp. 179-189 ◽  
Author(s):  
R WEIS ◽  
R LUITEN ◽  
W SKRANC ◽  
H SCHWAB ◽  
M WUBBOLTS ◽  
...  
Keyword(s):  
Cell Death ◽  
Yeast Cell ◽  
High Throughput ◽  
High Throughput Screening

Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death

2010 ◽  
Vol 407 (2) ◽  
pp. 205-210 ◽  
Author(s):  
Il Sang Yoon ◽  
Qingyan Au ◽  
Jack R. Barber ◽  
Shi Chung Ng ◽  
Bin Zhang
Keyword(s):  
Cell Death ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

scholarly journals High Throughput Screening Identifies a Novel Compound Protecting Cardiomyocytes from Doxorubicin-Induced Damage

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Szabolcs Gergely ◽  
Csaba Hegedűs ◽  
Petra Lakatos ◽  
Katalin Kovács ◽  
Renáta Gáspár ◽  
...  
Keyword(s):  
Cell Death ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antitumor Agents ◽  
Radical Scavenging ◽  
Drug Candidate ◽  
Necrotic Cell ◽  
Rat Cardiomyocytes ◽  
Scavenging Effect ◽  
Radical Scavenging Effect

Antracyclines are effective antitumor agents. One of the most commonly used antracyclines is doxorubicin, which can be successfully used to treat a diverse spectrum of tumors. Application of these drugs is limited by their cardiotoxic effect, which is determined by a lifetime cumulative dose. We set out to identify by high throughput screening cardioprotective compounds protecting cardiomyocytes from doxorubicin-induced injury. Ten thousand compounds of ChemBridge’s DIVERSet compound library were screened to identify compounds that can protect H9C2 rat cardiomyocytes against doxorubicin-induced cell death. The most effective compound proved protective in doxorubicin-treated primary rat cardiomyocytes and was further characterized to demonstrate that it significantly decreased doxorubicin-induced apoptotic and necrotic cell death and inhibited doxorubicin-induced activation of JNK MAP kinase without having considerable radical scavenging effect or interfering with the antitumor effect of doxorubicin. In fact the compound identified as 3-[2-(4-ethylphenyl)-2-oxoethyl]-1,2-dimethyl-1H-3,1-benzimidazol-3-ium bromide was toxic to all tumor cell lines tested even without doxorubicine treatment. This benzimidazole compound may lead, through further optimalization, to the development of a drug candidate protecting the heart from doxorubicin-induced injury.

scholarly journals Super-human cell death detection with biomarker-optimized neural networks

2020 ◽  
Author(s):  
Jeremy W. Linsley ◽  
Drew A. Linsley ◽  
Josh Lamstein ◽  
Gennadi Ryan ◽  
Kevan Shah ◽  
...  
Keyword(s):  
Neural Networks ◽  
Cell Death ◽  
High Throughput ◽  
High Throughput Screening ◽  
Morphological Characteristics ◽  
Ground Truth ◽  
Cell Types ◽  
Live Cells ◽  
New Paradigm ◽  
Cell Vitality

AbstractCell death is an essential process in biology that must be accounted for in live microscopy experiments. Nevertheless, cell death is difficult to detect without perturbing experiments with stains, dyes or biosensors that can bias experimental outcomes, lead to inconsistent results, and reduce the number of processes that can be simultaneously labelled. These additional steps also make live microscopy difficult to scale for high-throughput screening because of the cost, labor, and analysis they entail. We address this fundamental limitation of live microscopy with biomarker-optimized convolutional neural networks (BO-CNN): computer vision models trained with a ground truth biosensor that detect live cells with superhuman, 96% accuracy more than 100 times faster than previous methods. Our models learn to identify important morphological characteristics associated with cell vitality without human input or additional perturbations, and to generalize to other imaging modalities and cell types for which they have no specialized training. We demonstrate that we can interpret decisions from BO-CNN models to gain biological insight into the patterns they use to achieve superhuman accuracy. The BO-CNN approach is broadly useful for live microscopy, and affords a powerful new paradigm for advancing the state of high-throughput imaging in a variety of contexts.

scholarly journals Electroporation Knows No Boundaries

2015 ◽  
Vol 20 (8) ◽  
pp. 932-942 ◽  
Author(s):  
Christin Luft ◽  
Robin Ketteler
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Efficiency ◽  
Target Identification ◽  
Cell Types ◽  
Rnai Technology ◽  
Drug Target Identification ◽  
Interfering Rna ◽  
Calcium Phosphate Precipitation

The discovery of RNA interference (RNAi) has enabled several breakthrough discoveries in the area of functional genomics. The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms. One of the major hurdles for the use of RNAi in high-throughput screening has been delivery to cells and tissues. Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means. Electroporation is a powerful and versatile method for delivery of RNA, DNA, peptides, and small molecules into cell lines and primary cells, as well as whole tissues and organisms. Of particular interest is the use of electroporation for delivery of small interfering RNA oligonucleotides and clustered regularly interspaced short palindromic repeats/Cas9 plasmid vectors in high-throughput screening and for therapeutic applications. Here, we will review the use of electroporation in high-throughput screening in cell lines and tissues.

A Fluorescence-Based High-Throughput Screening Assay for the Identification of T-Type Calcium Channel Blockers

2009 ◽  
Vol 7 (3) ◽  
pp. 266-280 ◽  
Author(s):  
Francesco Belardetti ◽  
Elizabeth Tringham ◽  
Cyrus Eduljee ◽  
Xinpo Jiang ◽  
Haiheng Dong ◽  
...  
Keyword(s):  
Calcium Channel ◽  
High Throughput ◽  
Calcium Channel Blockers ◽  
High Throughput Screening ◽  
Channel Blockers ◽  
Screening Assay ◽  
High Throughput Screening Assay

scholarly journals Droplet-Based Microfluidics

2013 ◽  
Vol 19 (4) ◽  
pp. 483-496 ◽  
Author(s):  
Oliver J. Dressler ◽  
Richard M. Maceiczyk ◽  
Soo-Ik Chang ◽  
Andrew J. deMello
Keyword(s):  
Biological Sciences ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Efficiency ◽  
Small Sample ◽  
Operational Flexibility ◽  
High Throughput Experimentation ◽  
The Impact ◽  
Functional Components ◽  
Fabrication Costs

Over the past two decades, the application of microengineered systems in the chemical and biological sciences has transformed the way in which high-throughput experimentation is performed. The ability to fabricate complex microfluidic architectures has allowed scientists to create new experimental formats for processing ultra-small analytical volumes in short periods and with high efficiency. The development of such microfluidic systems has been driven by a range of fundamental features that accompany miniaturization. These include the ability to handle small sample volumes, ultra-low fabrication costs, reduced analysis times, enhanced operational flexibility, facile automation, and the ability to integrate functional components within complex analytical schemes. Herein we discuss the impact of microfluidics in the area of high-throughput screening and drug discovery and highlight some of the most pertinent studies in the recent literature.

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