Why are there current fluctuations in herg patch clamp assay using port a patch nanion. Electrophysiological analysis of mammalian cells expressing herg. That being said, although the benefits of automation seem obvious at face value, in practical experience it is seldom the case that a wellbehaved manual patch clamp assay with mammalian cells. Both filtration and homogeneous flashblue herg binding assay have been optimized with 125 ibekm1 panels 711. In addition, fluorescence material is safer and less expensive than radioactive material for receptor binding or chromium release cytotoxicity assays. The assay is based on the principle of fluorescence polarization, where a redshifted fluorescent tracer displays a high polarization when bound to the herg channel and a low polarization. Four representative traces from the same cell are superimposed to visualize the effect of increasing concentrations of the known.
Implementation of a kinetic herg assay protocol using qpatch. Manual wholecell patchclamping of the herg cardiac k channel. The advantage of whole cell patch clamp recording over sharp electrode technique recording is that the larger opening at the tip of the patch clamp electrode provides lower resistance and thus better electrical access to the inside of the cell. As neurons derived from axolnscs mature over time, the number of cells spiking increased up to 100% of the total number of neurons recorded at one month after plating figure 3a. Validation of choherg cell line was carried with whole cell patch clamping to show faimily of tail currents, iv relationships, and ic50 determinations of two positive blockers figures 14. To identify potential herg liabilities early in drug discovery programs and thus avoid problems with herg channel interactions for latestage compounds, it has become common practice in drug discovery. Test substance solution is flowed into narrow channel by laminar action to. Introduction the whole cell patch clamp technique using cell lines transfected with human etheragogorelated gene herg has become more important recently for prediction of a potential risk of qtinterval prolongation that. Fluorescence alternatives to popular biological assays. In this study, hek 293herg cells merck millipore, billerica, ma, usa, which are hek 293 cells stably expressing herg, and choherg cells merck millipore, which are cho cells stably expressing herg, were used. There are several established but indirect methods for evaluation of herg liability that are amenable to higherthroughput techniques. Apr 27, 2017 creative bioarray uses the stateoftheart automated qpatchht system to provide a higherthroughput herg safety assay with better consistency at a lower cost. Whole cell patch clamp was used to record currents at 37c.
Bsys currently operates 3 automated patch clamp devices sophion qpatch. The intercellline reproducibility of herg assay using. Wholecell configuration of the patchclamp technique in the herg channel assay to predict the ability of a compound to prolong qt interval article in. Patch clamp technique is the gold standard for carrying out. Electrophysiological analysis of mammalian cells expressing. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or longterm exposure on the amplitude of ikr in human embryonic kidney 293 hek293 cells stably transfected with the herg channel wholecell. Why are there current fluctuations in herg patch clamp assay. An in vitro electrophysiological assay system, which can assess compound. Sep 30, 2010 this testing is used, as part of a larger battery of tests, to help predict the cardiac safety profile of a drug. Validation of cho herg cell line was carried with whole cell patch clamping to show faimily of tail currents, iv relationships, and ic50 determinations of two positive blockers figures 14.
For me with cell line run down is huge, in 5 min time 50. Creative bioarray uses the stateoftheart automated qpatchht system to provide a higherthroughput herg safety assay with better consistency at a lower cost. Biochemical herg assays thermo fisher scientific us. Patchclamp assay from single neuron down to single channel, thanks to the different recording configurations wholecell, perforatedpatch, cellattached, outsideout, patchclamp techniques provide the highest resolution for electrophysiological recordings in ion. Automated patch clamp assays have been successfully used in a. For some types of cells, it is convenient to apply both methods simultaneously to break the patch. Hts screening of herg blockers using 2ndgeneration. Wholecell patchclamp voltageclamp experiments were performed on. This method is considered the gold standard in regards to testing ion channels. Comparison of the predictor herg fp assay to the patch clamp method has previously been shown to correlate very closely. This testing is used, as part of a larger battery of tests, to help predict the cardiac safety profile of a drug. Characterization of a herg screen using the ionworks ht.
A patchclamp investigation of hek 293 cells or cho cells expressing herg was performed in an automated way by using a 384wellpatchclamp system. It also provides typical voltage protocols used for assessing the basic electrophysiological properties of these currents and for assessing the effects of compounds on herg tail currents. We also show that the sensitivity of the assay can be improved by adding a surfactant to. Panel c shows a family of herg currents from an experiment described above. The qpatch htx and the syncropatch 384pe systems deliver high quality. The herg safety service is performed at our parent company, evotec, and is a cell based assay which employs the qpatch htx system sophion bioscience as or the syncropatch 384pe nanion technologies as automated patch clamp electrophysiology measurements. The metrion biosciences team recently produced an application note reporting the successful validation of the milnes voltage protocol for herg screening using a qpatch 48 automated patch clamp apc assay platform. Sep 02, 2014 while this article focuses on herg channel recordings using the qpatch sophion as, copenhagen, denmark technology, many of the assay and protocol details given in this article can be transferred for setting up different ion channel assays by automated patch clamp and are similar on other planar patch clamp platforms. I am able to achive whole cell stage with 1gohm seal upto 20 min and. The 3hdofetilide binding assay is a predictive screening.
Money will be saved by not buying specialized equipment for each assay. The voltage clamp was difficult both teoretically and practically, not a lot of people could have experience doing that. Implementation of a kinetic herg assay protocol using. Conventional patch clamping, now also referred to as manual patchclamp to distinguish it from the recently developed automated patchclamp described below see section vi, is accomplished by sealing the small tip of a pipette to the surface of the cell membrane in such a way that is possible to isolate a tiny membrane area patch from the rest of the membrane. Measured on the syncropatch 384pe using whole cell patch clamp methodology and multihole chips 4 holes per well. Why are there current fluctuations in herg patch clamp. Wholecell patch clamp can be used to characterize the maturation of neuronal cultures, both at the level of individual cells and at the networks connectivity level. Early identification of herg liability in drug discovery. The temperature of the systems cell hotel was set to. This method is considered comparison of the predictor herg fp assay to the patch clamp method has previously been shown to correlate very closely. Screenshots of the patchcontrol 384 software showing herg current traces in response to the cipa voltage step protocol. Patch clamp technique, awarded nobel prize, has originally 4 different configurations, one of which is called a whole cell configuration.
However, it is often seen that the responsible scientist from the biotech company is not familiar with patch clamp, or. Wholecell configuration of the patchclamp technique in. Dec 16, 2015 a patchclamp investigation of hek 293 cells or cho cells expressing herg was performed in an automated way by using a 384wellpatchclamp system. Conventional patch clamping, now also referred to as manual patchclamp to distinguish it from the recently developed automated patchclamp described below see section vi, is accomplished by sealing the small tip of a pipette to the surface of the cell membrane in such a way that is possible to isolate a tiny membrane area patch from the rest of the membrane and to control its. Fhnd004 is a newly synthesized epidermal growth factor receptor egfr inhibitor for the treatment of nonsmall cell lung cancer nsclc. Family of tail currents from whole cell patch clamping of cho herg cells fig.
The herg safety service is performed at our parent company, evotec, and is a cellbased assay which employs the qpatch htx system sophion bioscience as or the syncropatch 384pe nanion technologies as automated patch clamp electrophysiology measurements. Wholecell configuration of the patchclamp technique in the. The gold standard manual whole cell patch clamp is used for compound profiling in. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or longterm exposure on the amplitude of ikr in human embryonic kidney 293 hek293 cells stably transfected with the herg channel whole cell configuration of the patch clamp technique.
Time will be saved compared to whole cell caco2 assays and patch clamp herg binding assays. Ionic current measurements request a study outline from ipst wholecell current amplitude and kinetics measurements verify the results of the interaction of a test article with a selected ionic current. This video describes the details of patch clamp technique starting from very basics and the utility of this technique in neuroscience. Inhouse and commercially available stem cell derived cells. Wholecell patch clamp recording was performed on wild type wt, mutant.
A set of 70 compounds, including herg antagonists with potencies spanning 3 orders of magnitude, were tested on herg302hek cells using protocols aimed at either. That being said, although the benefits of automation seem obvious at face value, in practical experience it is seldom the case that a wellbehaved manual patchclamp assay with mammalian cells. Wholecell patchclamp voltageclamp experiments were performed on chinese hamster ovary cho cells stably transfected with herg1a kv11. I am doing herg voltage clamp protocol using port a patch but i am unable to get a.
Comparison of the predictor herg fp assay to the patch clamp method. Manual wholecell patchclamping provides the most sensitive and accurate way to examine the biophysical and pharmacological. For small number of compounds or compounds identified with qpatch, we can also perform conventional whole cell patch clamp assay to get detailed mechanistic information. Clamp technique in the herg channel assay to predict the ability of a compound to prolong qt interval. Patch clamp technique, awarded nobel prize, has originally 4 different configurations, one of which is called a wholecell configuration. For the biotechs who are not familiar with herg assay. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patch clamp electrophysiology studies. Invitrogens predictor herg fluorescence polarization. Pdf early identification of herg liability in drug discovery. Currently, the conventional wholecell patch clamp assay is the most reliable method available to. Whole cell configuration of the patch clamp technique in the herg channel assay to predict the ability of a compound to prolong qt interval. Wholecell recordings or singlechannel analysis in various expression systems hek 293, cho, neuroblastoma etc. Patch clamp technique is the gold standard for carrying.
The inter cell line reproducibility of herg assay using the whole cell patch clamping 1. However, it is often seen that the responsible scientist from the biotech company is not familiar with patch clamp, or voltage clamp with single electrode. Aug 12, 2018 this video describes the details of patch clamp technique starting from very basics and the utility of this technique in neuroscience. The flyscreen machine uses glass micropipettes similar to conventional patch clamp pipettes. Wholecell configuration of the patchclamp technique in the herg. This chapter describes how to record herg currents in a recombinant cell line using the whole cell patch clamp technique. To make the assay more translatable to clinical studies, we have conducted whole. Introduction the wholecell patchclamp technique using cell lines transfected with human etheragogorelated gene herg has become more important recently for prediction of a potential risk of qtinterval prolongation that. Automated patch clamp an overview sciencedirect topics. Traditionally herg screening is performed with a whole cell voltage patch clamp apparatus. Wholecell configuration of the patchclamp technique in the herg channel assay to predict the ability of a compound to prolong qt interval. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patchclamp electrophysiology studies.
The intercellline reproducibility of herg assay using the wholecell patchclamping 1. It includes a current clamp and a voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc. The intercellline reproducibility of herg assay using the. Dec 16, 2015 cell culture and cell preparation for patch clamp analysis. The gold standard to carry out the glp herg assay is the technique so called patch clamp voltage clamp method wholecell configuration. However, the glass micropipettes are inverted placing the cell inside for sealing. In the automated patchclamp measurements performed here, kv currents. This chapter describes how to record herg currents in a recombinant cell line using the wholecell patchclamp technique. Invitrogens predictor herg fluorescence polarization assay. Biochemical herg assays thermo fisher scientific ru. May 31, 2018 fhnd004 is a newly synthesized epidermal growth factor receptor egfr inhibitor for the treatment of nonsmall cell lung cancer nsclc. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or long. Patch clamp electrophysiology in a defined physiological saline solution pss represents the standard method for assaying drug effects on the channel.
In this study, hek 293 herg cells merck millipore, billerica, ma, usa, which are hek 293 cells stably expressing herg, and cho herg cells merck millipore, which are cho cells stably expressing herg, were used. The impact of automated patch clamping apc on ion channel screening is now. Automated patchclamp methods for the herg cardiac potassium. Identifying modulators of herg channel activity using the.
Why are there current fluctuations in herg patch clamp assay using. While the patch clamp method works very well for identifying compounds that block potassium ion flow through. The herg safety service is performed by our parent company, evotec, and is a cellbased assay which employs the qpatch htx system sophion bioscience as or the syncropatch 384pe nanion technologies as automated patch clamp electrophysiology measurements. Cell culture and cell preparation for patchclamp analysis.