The results demonstrate that QPatch 16 can provide high-quality whole-cell current data from fast voltage-activated Na+ channels, and that protocols can be defined that allow analysis of drug potency (IC50), inactivation kinetic properties, as well as state- and frequency-dependency of drug action.

• High fidelity voltage and current clamp recordings of Cor.4U in physiological solution
• Demonstration of the presence of I_Na, I_Ca as well as I_Kr
• The use of fluoride in the internal solution resulted in lower
  I_Ca and shortened action potential durations
• Paced and spontaneous action potentials
• Pharmacological agents show expected effects on action
  potentials in Cor.4U

The new adaptive protocol enables increased control of the state that voltage-gated channels are in during an experiment, leading to reduced variability of data and increased confidence in compound testing results, on a 384-well high throughput automated patch clamp platform.

This report summarizes the experiments that were done running fast ligand-gated assays on QPatch 48X. QPatch 48X in multi-hole mode patches 10 cells per well instead of just one which has been shown to increase the overall success rate due to the fact that cells with less or no expression of the ion channel of interest will be patched in parallel with other cells that has better expression. The currents from all 10 holes are thus added together and therefore gives rise to increased current amplitudes from each well. The 10 holes obviously take up more space in the flow channel in the QPlate chip and we were curious to know if these changes had any effect on the measured signal from the ten cells. We set out to test different ligand-gated ion channels that are well known and already validated on QPatch HT to determine if the signal onset was comparable to QPatch 48X in multi-hole mode. Furthermore, we investigated the effect of known compounds: agonists, antagonist and modulators to see if any changes in data quality could be detected.

CHO cells expressing the cold-sensitive ion channel TRPM8 were tested on the QPatch, and HEK cells expressing TRPM8 were tested on QPatch HTX. Two agonists of TRPM8 were used for EC₅₀ determinations: menthol and icilin. All data presented was obtained with QPatch and analyzed with QPatch Assay Software

TMEM16A (ANO1) is a Ca2+-activated Cl-channel (CaCC) that is involved in a plethora of physiological and pathophysiological conditions. The channel was suggested as target for treatment of asthma, secretory diarrheas, and hypertension. TMEM16A is unique as its gating ynergistically depends on voltage and cytosolic Ca2+ (Scudieri et al. 2012). A robust TMEM16A assay was long awaited but progress was hampered as many automated patch clamp devices rely on the use of fluoride in the internal solution. Fluoride exhibits a very low solubility with calcium which means that calcium that is added to the internal solution to activate the channel has a strong tendency to precipitate, making it impossible to correctly adjust the calcium concentration.

Ca2+-activated K+ channels (KCa) are integral players in setting the membrane potential and regulating Ca2+ homeostasis. The human genome consists of a total of 8 KCa channels that differ in their unitary conductance, genetic relation to each other and mechanism of Ca2+ binding. The channels are subdivided in two groups according to the aforementioned characteristics. The first group was initially characterized by their structural similarity and comprises of KCa1.1 (BK), KCa4.1, KCa4.2 and KCa5.1. Later studies revealed that these channels have in fact a different activation mode and only KCa1.1 (BK) is a truly Ca2+-activated channel aside from its voltage-dependence. The second group is characterized by their unitary conductance and encompasses three small conductance (SK) channels KCa2.1 (SK1), KCa2.2 (SK2) and KCa2.3
(SK3), and an intermediate-conductance channel KCa3.1 that is also known as SK4, IKCa1, IK-1 or KCa4 encoded by KCNN4 (Wei et al., 2005). KCa3.1 was cloned independently by three groups in 1997, therefore, the channels biophysical and pharmacological properties are already well-established (Wulff & Castle, 2010).

To better understand how compounds function in warm-blooded animals such as humans, it is key to be able to test compound potency at physiological temperature. Our automated patch clamp systems provide real time measurement of temperature, and thus the effect of temperature change. Temperature regulation is done directly at the measurement site, and not by thermal control of the whole system. Four different compounds were tested in a dose-response format on Qube 384 at differing temperatures. All four compounds showed increased effect at elevated temperature compared to room temperature. Qube is suitable for screening compounds at temperatures from 8° C to 40° C (or 8° C above dew point).

The new adaptive protocol enables increased control of the state that voltage-gated channels are in during an experiment, leading to reduced variability of data and increased confidence in compound testing results, on a 384-well high throughput automated patch clamp platform.

The aim of this study is to observe the consistency of a current signal (elicited by an agonist or a voltage change) before and after antagonist or blocker application. This report presents data monitored on the automated QPatch 16 based on whole-cell current recordings from ligand-gated ion channels (LGIC); the glutamate-activated ligand-gated ion channels Glu5R expressed in HEK293 cells from Neurosearch and GluR6 (GRIK2) from Millipore as well as acid-sensing ion channel 3 (ASIC3) expressed in HEK293 cells. Similar experiments were performed on voltage-gated ion channels (VGIC); HEK293-Na_v1.2a and CHO-hERG.

The ATP-gated P2X₂ and P2X₃ receptors are cation-permeable
ligand-gated ion channels that open in response to extracellular adenosine 5′-triphosphate (ATP) binding. They are widely expressed in the body, including the peripheral nervous system, making them an interesting target against pain^1–3.

When P2X₂ and P2X₃ receptors are expressed together, the functional receptors will be trimeric³ and function either as P2X_2/3 heteromers, P2X₂ homomers, or P2X₃ homomers. The ratio between P2X₂ and P2X₃ homomers and P2X_2/3 heteromers vary from cell to cell, so to limit the variation between sites, only multihole QChips with ten patch holes per site are used in the assays.

P2X₂ and P2X₃ receptors display fast and extensive desensitization, making assays vulnerable to rundown due to poor compound washout or insufficient recovery. Here we show an assay with high stability, high success rates, and low rundown.

The voltage-gated potassium channel K_V2.1 is the main contributor to the delayed rectifier potassium current in neurons and in the cortex (Du et al. 1998, Murakoshi et al. 1999). It has been suggested that K_V2.1 activation confers neuroprotection in a state of neuronal hyperexcitability via hyperpolarizing the membrane potential (Du et al. 2000, Misonou et al. 2005, Misonou et al. 2005a). Therefore, K_V2.1 may be an attractive target in epilepsy research. Also, the ion channel has previously been shown to play a central role in neurodegeneration, ageing and pancreatic β cell signaling.

Recommended cell concentration on both QPatch ll and Qube depends on both assay complexity, assay longevity and especially on cell quality. We generally recommend having a cell density of 3×10⁶ cells/ml on both Qube and QPatch ll. However, the cell consumption for both systems can be reduced significantly, with a factor of at least 6, and still ensure success rates above 90%. We recommend that optimal cell concentrations are evaluated when working with cells that are expensive or in short supply. Furthermore, QPatch ll can now be loaded with as little as 50 μl cells for a single experiment.

It has been shown that the human muscle cell line TE671 has an endogenous nicotinic acetylcholine alpha 1 receptor (nAChRα1) (Morris et al,. 1991). TE671 cells have therefore been used for experiments with nAChRα1 as a target. This brief report describes the experimental set-up of TE671 and nAChRα1 on the QPatch.

Using the optical features of Qube Opto 384, it is possible to evaluate both light-activated ion channels and photoactivated ligands. Here we outline a few of the studies we have performed on Qube Opto 384.

Channelrhodopsin 2 (ChR2) could be activated and the channelrhodopsin 2 mediated current could be manipulated in both a light and voltage-dependent manner, with activation times as short as 4 ms. A light-induced chloride current could also be elicited, employing the chloride-conducting channelrhodopsin iC⁺⁺.

Caged γ-aminobutyric acid (Rubi-GABA) could be activated by light and give rise to a GABA_AR mediated current. The GABA response was both concentration and the light intensity dependent. The optical activation of GABA combined with microfluidic channels also enabled ultra-short ligand exposure times.

The QPlate is the biochip applied for electrophysiological measurements using the QPatch automated patch clamp (APC) instrument. The unique QPlate design ensures high and consistent experiment quality and secures several vital properties for high-throughput patch-clamp, including:

– Giga-ohm seal formation in physiological solutions

– Minimal compound adsorption

– Fast and efficient (100%) liquid exchange

– Maintenance-free, single-use electrodes

– Several QPlate configurations to accommodate different experiment- and throughput requirements

– A non-compromised testing environment

Native cells represent a promising alternative to cell lines due to more physiological behaviour.

Primary brain neurons and astrocytes were tested on Qube 384

• Neuronal Na+ and K+ currents were measured in voltage clamp
• Neuronal action potentials were measured in current clamp
• Astrocyte K+-currents were elicited with both ramp- and step-protocols

Studies of GABA_A ion channels using the automated patch clamp platform Qube 384 with focus on:

• Short ligand exposure with repetitive stimulations with EC₅₀
  concentrations of GABA
• Effects of agonists, antagonists and modulators
• Cumulative and non-cumulative concentration-response relationships
• Characterizing the pharmacological properties of four cell lines expressing different GABA_A subtypes

In patch clamp recordings cell dialysis of intracellular constituents can be minimized by employing perforated whole-cell formation.
This method allows electrical access to the cell while maintaining the integrity of the majority of cytoplasmic components. Here we report how to perform perforated patch clamp recordings on Qube 384 with or without the use of intracellular (IC) solution exchange (IC exchange).
• Recommendations for perforated patch clamp experiments with or without using IC exchange
• Comparison of four perforating agents (seal resistance, current level and success rate)
The first part of this report consists of a fast guide for using perforated patch on Qube 384, followed by an in-depth run-through of the experiments underlying our recommendations

NIH/3T3 is a mouse embryonic fibroblast cell line. NIH/3T3 cells are established from NIH Swiss mouse embryos. These cells are highly contact inhibited and are sensitive to sarcoma virus focus formation and leukaemia virus propagation. These cells have now lost their contact inhibition. Contact inhibition is the natural process of arresting cell growth when two or more cells come into contact with each other. The established NIH/3T3 line was subjected to more than 5 serial cycles of subcloning in order to develop a subclone with morphologic characteristics best suited for transformation assays. It is therefore used for DNA transfection studies.
Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a chloride channel and controls the regulation of other transport pathways. Mutations in the CFTR gene have been found to cause cystic fibrosis (CF) and congenital bilateral aplasia of the vas deferens (CBAVD). The ΔF508CFTR mutation of the CFTR gene was stably transfected into the NIH/3T3 cell line and used for functional studies on QPatch.

As an alternative to standard pharmacological procedures for Na_V1.5(Late) assays, we present a more reliable and accurate Na_V1.5(Late) assay on QPatch that removes the requirement for activators like veratridine and ATX-II and delivers improved cardiac safety screening reliability and cost.

• High fidelity QPatch recordings of small amplitude Na_V1.5 (Late) currents from Long QT3 syndrome mutant channels.
• Stable recordings without pharmacological activators allow more reliable drug potency assessment.
• Pharmacologically validated with sodium channel blockers with a preference for the Na_V1.5(Late).

The sodium channel Na_V1.1, highly expressed in fast spiking interneurons (FSIs), initiates the interneuron’s action potentials and promotes γ-oscillations, which are important for memory encoding and other cognitive functions. An impaired function of FSIs is associated with disorders like autism, schizophrenia, Alzheimer’s and others. Potentiators of Na_V1.1 can restore γ-oscillations by recovering reduced FSI function and they have been shown to minimize cognitive dysfunction in transgenic mice with decreased levels of Na_V1.1 in parvalbumin-positive neurons.

Studies on GABAA ion channels using the automated patch clamp platform QPatch with the focus on:

• Effects of agonists, antagonists, and modulators
• Concentration-response relationships
• EC₅₀ and IC₅₀ determination
• Characterizing both the pharmacology of a specific isolated GABAA subtype and the physiological GABA response of cultured rat astrocytes

It was possible to obtain both manual and automated patch clamp (APC) recordings of the labile and scarce Wnt proteins (350-400 AA, 35-45 kDa) after thorough optimisation of compound handling.

Here we highlight aspects of handling of Wnt proteins optimised for APC testing, which could also be applied to other large molecules (e.g. toxins, peptides, antibodies).

Qube 384 is capable of performing pharmacology measurements with large molecules.

Low volume microfluidic application ensures that expensive and scarce molecules like antibodies and toxins can be efficiently tested.

Key pharmacology metrics defined in these assays are comparable to values reported in the literature.

To determine if engineered IgG anti-α-cobratoxin antibodies would prevent the blocking of the nicotinic acetylcholine receptor, a protocol was set up using QPatch II. Here we show that:

• QPatch II is well suited for efficiently characterizing both peptide toxins and antibody function.
• Current induced by adding acetylcholine could be abolished completely using α-cobratoxin, and that this effect could be reversed by preincubating the toxin with different IgGs before injection.
• Prevention of current inhibition by the IgGs was dose-dependent, and the potency of different IgGs could be determined using this method.
• Increased affinity between the IgGs and α-cobratoxin resulted in higher neutralization potency.

CiPA recommended Ca_V1.2 assay protocols were run on Qube
384 using Ready-To-Use (RTU) Ca_V1.2 cells from NMI TT with
~80% success rate, low run-down and reliable pharmacology.
We demonstrate the quality and versatility of ready-to-use Ca_V1.2 cells, but also that RTU cells can be a valuable addition to the laboratory tool pack to 1) decrease variation from cell culture and 2) decrease dependence on skilled cell culture personnel.

• Ready-to-use cells easily ignites your experiment within
minutes
• Up to 82% success rate
• Reliable pharmacology including safety pharmacology
recommended by CiPA
• Rundown of only +0.2% ± 0.06% per minute

Internal addition of compounds targeting the chloride channel CIC-1. A robust assay with good pharmacology and high Z-score

• A robust assay of ClC-1 with Z’ constantly >0.5
• Biophysical and pharmacological characteristics as expected for ClC-1
• Qube offers the possibility to apply compounds both intra- and extracellularly
• Advanced analysis of compound effects on channel kinetics are available at a few click

In this report, we show a ClC-1 assay on Qube that shows biophysical characteristics as expected for ClC-1. We further introduce Sophion`s Analyzer software that allows rapid analysis of large data sets to answer advanced electrophysiological questions, in the present case: What is the mode of action of a novel, unknown compound?

Rat basophilic leukaemia (RBL) cells endogenously express calcium-release-activated-calcium (CRAC) channels (1). CRAC channels are activated by depletion of intracellular calcium stores, probably via the involvement of STIM-1 (stromal interaction molecule) sensing the depletion of the stores and travelling to the cell membrane activating the channel (2, 3). Several approaches can be used to deplete calcium stores ultimately leading to activation of I_CRAC and most of these have been tried on QPatch. This document describes the results of these experiments and discusses the best conditions for studying I_CRAC with QPatch.

• High fidelity voltage and current clamp recordings of human iPS cell-derived cardiomyocytes (Cor.4U^®) on Qube
• Demonstration of the presence of I_Na, I_K and I_Ca
• High throughput current clamp recordings
• Pharmacological characterization of action potentials and isolated currents
• Using Sophion Analyzer software it is possible to easily extract key parameters relevant for studying compound effects on action potentials

The Na_V1.5 channel is critical in maintaining the physiological electrical function in cardiomyocytes, as it is the heart’s main depolarising current. Here we focus on the Na_V1.5 channel in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an important target for cardiac-drug and safety screening.

• The hiPSC-CMs Na_V1.5 compound screening was performed with assay success rate (quality filters) up to 60% and ~ 20 useful experiments per 48 recording site plate.

• The study demonstrates that it is possible to screen compounds on hiPSC-CMs using the QPatch II.

• The experimental success rate of hiPSC-CMs assays was variable depending on the quality and maturation of the cell population.

In summary, it is possible to perform measurements on QPatch II of hiPSC-CMs in physiological ringers with voltage-gated assays yielding up to 50% success rate, depending on the ion channel target. These measurements demonstrate a heterogeneous expression of the cardiac ion channels, which consequently lowers the success rates (up to 20%) of action potential measurements. As 1) the hiPSC-CM quality and maturity increases and 2) cell suspension preparation is further optimized, we expect the throughput of these measurements to increase. Eventually, we envision that APC can be used as a characterization tool to assist the continuous development of hiPSC-CMs as well as for cardiac drug screening and disease modelling.

• Constantly high success rates (99% in 3 experiments)
• Giga-ohm seal resistances in almost all wells
• Very low data spread
  • All CV values of DMSO control below 10%
  • No false positives with cutoff criteria 3 X SD
  • Z’ constantly greater 0.75
• Discrimination between different modes of inhibition (state-dependency)
• Up to 384 individual IC₅₀ values (3072 data points) determined in just one experiment
• Using Qube in combination with Stacker it would be possible to determine up to 7680 IC₅₀ (60,000 data points) per 24 hours

Large molecules: evaluating the neutralization potential of recombinant antibodies against snake neurotoxins

Qube 384 is capable of performing pharmacology measurements with large molecules.

Qube 384 provides high throughput and high-quality recordings, enabling the evaluation of neurotoxins and neutralizing antibodies in an assay with a success rate of 95%.

K_V1.5 channels conduct the ultra-rapid delayed rectifier current (I_Kur) that contributes to action potential repolarization of the heart. Mutations in the encoding gene (KCNA5) have been related to both atrial fibrillation and sudden cardiac death. K_V1.5 is also found in other tissues, such as vascular smooth muscle, CNS, microglia, and Schwann cells (Clarkson et Al., 1998).

The pharmacological blockade of K_V1.5 has been suggested as a prevention and treatment of various arrhythmia (Karczewski et al., 2009) In the present study, we present an assay suitable for a high throughput screening campaign.

In this study, by using our QPatch II system and the temperature control feature, we report a significant difference in hERG kinetics at 22°C and 35°C. By testing four hERG blockers, we were able to measure channel activity and investigate compound effects at the two temperatures.
IC50 values obtained from the compounds tested on the QPatch II are similar to those obtained on the Qube 384 and agree with literature.

The QPatch 16 automated patch clamp system was used to establish and record currents from HEK293 cells stably expressing TRPV1 channels.

TRPV1 response is stable on the QPatch platform and it is easy to perform agonist and antagonist studies. The half maxima effective and inhibitory concentrations (EC50 and IC50) found in this study are comparable to literature values.

Performing automated patch clamp experiments with different intracellular solutions is somehow difficult. Using QPatch with its multi well reservoir is it possible to test eight different solutions on the same cell line. In this study the multi well reservoir has been used for experiments on the voltage-dependent large conductance Ca2+-activated K+ channels, often referred to as BK channels. A human embryonic kidney cell line (HEK293) expressing the BK channel was used.
BK channels are large conductance calcium and voltage gated potassium channels, which allow potassium fluxwhen activated with intra cellular calcium and/or membrane potential (Strøbæk et al., 1996).

It is common that potential customers ask Sophion Bioscience to use QPatch to test some of their compounds on specific ion channels before buying a system. In this application report, we present some of the results obtained from a validation study done for a pharmaceutical company. Currents from the N-type
voltage-gated calcium channels (Ca_V2.2) were studied using
QPatch. A blocker of the Ca_V2.2 current, compound A, was
applied in increasing concentrations to study the dose-response
effects and estimate IC₅₀ values. Furthermore, flunarizine which is known to block T-type Ca²⁺/Na⁺ channels and have low potency on N-type Ca channels, was tested.

hKir2.1 exhibit strong inward rectification with major current activity at very negative potentials. The channel has a fundamental role in controlling and maintaining the resting membrane potential. hKir2.1 is encoded by the KCNJ2 gene and mutation in this gene can cause cardiac arrhythmia.

The QPatch16 automated patch clamp system was used to establish and record currents from HEK293 cells stably expressing Ca_V3.2 channels. Whole cell currents were measured upon application of a voltage-step protocol to obtain I-V curves.

The development of screening assays for the Ca_V1.2 channel has been challenging due to the tendency of the channel to exhibit declining current levels (rundown) during the experiment. Here we report a robust Ca_V1.2 assay yielding high success rates, low rundown and reliable pharmacology.

Pharmacology and current-voltage relationship in accordance with literature values

Success rates of up to 98%

Stable currents with rundown as low as 2% per min

The development of screening assays for the Ca_V1.2 channel has been challenging due to the tendency of Ca_V1.2 expressing cell lines to exhibit declining current levels (rundown) during the experiment^1,2. Here we report a robust Ca_V1.2 assay yielding high success rates, low rundown and reliable pharmacology.

Pharmacology and current-voltage relationship in accordance with literature values

Success rates up to 94%

Stable currents with rundown as low as (1.2 ± 0.9)% per minute

The CiPA protocol yields stable currents with rundown as low as (1 ± 1)% per minute

This study presents results produced on the QPatch with GH4-C1 cells expressing the homomeric nicotinic ligand gated α7 ion channel (nAChR α7). nAChR α7 is linked to many normal and pathological conditions. It is important in memory formation, arousal, and attention, nicotine addiction, and nicotine-induced reversal of age-related memory deficits. nAChR α7 activation leads to neuroprotection against amiloid-β-induced neurotoxicity, which suggests that the receptor may play an important role in Alzheimer’s disease. Also their role in hippocampal auditory gating has linked the receptor to schizophrenia. These findings makes this particular channel an interesting target for pharmaceutical
companies. The fast kinetics of the α7 channel makes this a difficult ion channel to work on even in conventional patch clamp rigs. At Sophion we have developed assays that captures this current in a reproducible way enabling automated technology to be used in the search for blockers and positive allosteric modulators of this channel.

Series resistance compensation for fast activating ion channels can help remove voltage clamp errors and increase clamp accuracy. All Sophions patch clamp amplifiers utilize a patented technique enabling up to 100% RS compensation. In addition, Sophion amplifiers are equipped with automatic clip detection to avoid loss of cells due to fatal oscillations, thereby increasing data throughput while maintaining high quality recordings. Here we show that voltage clamp errors can be up to 20-30 mV when uncompensated and in general that errors occur if not using RS compensation. However, we also show that the increase of accuracy due to use of RS compensation comes with a price of a lower success rate, and that the settings are critical to maximize success rates while avoiding or decreasing errors. Last we demonstrate the importance of automated clip detection.

• The nicotinic acetylcholine receptor alpha1 (nAChR α1) is a fast desensitizing channel that makes the development of a stable high throughput assay on automated patch clamp systems challenging.
• The solution stacking feature on Qube reduces ligand exposure times below 1 s. Using this feature, it is now possible to evoke stable acetylcholine signals for agonist concentrations at least up to EC₅₀.
• An assay for nAChR α1 endogenously expressed in TE671 cells with 5 µM acetylcholine showed the following characteristics:
  • Overall success rate of 89%
  • Overall whole-cell seal resistance = 1.2 ± 0.4 GΩ (SD)
  • Coefficient of variance for rows and columns in DMSO control ≤12%
  • IC₅₀(max/min)(tetracaine) of a non-cumulative concentration response experiment = 1.5
  • Z’ greater than 0.70
• The assay stability is also owed to unique architecture of the QChip where microfluidic channels ensure rapid and complete solution exchange of compound and agonist.

Human-induced pluripotent stem cells (hiPSCs) were developed a decade ago and hold great promise for disease modelling, drug discovery and personalized medicine, especially in cardiac and neurological diseases1. Dysfunctional ion channels are one of the major targets in these diseases and thus, as the iPSC technology improves, a requirement arises for instrumentation that can characterize electrophysiological properties of iPSCs in a high-throughput fashion. Here, we demonstrate the use of the automated patch clamp (APC) platforms Qube 384 and QPatch in three characterization studies of hiPSC motor neurons derived from patients and healthy individuals. The results include:

APC recording of hiPSC-derived motor neurons with high success rates on Qube (60%) and QPatch (30%)

Biophysical characterization of voltage-gated channels, Na⁺ (Na_V) and K⁺ (K_V), in healthy hiPSCs

Measurements of a ligand-gated channel (GABA_A receptor) in healthy control cells

Screening of hiPSC motor neurons derived from Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) patients

Comparison of hiPSC motor neurons derived from two different healthy individuals

Cardiac safety side-effects remain the leading cause of new compound attrition during the drug discovery process (Valentin and Redfern, 2017), suggesting that more robust preclinical in vitro, ex vivo and in vivo assays and models are required to predict human clinical risk. Currently, the industry is moving away from an over-reliance on the human Ether-a-go-go related potassium channel (hERG, KV11.1) assay and QT prolongation readouts by developing new initiatives that provide a more balanced assessment of patient risk, focusing in particular on cardiac arrhythmia liability. The FDA’s Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative and Japanese regulatory authorities (JiCSA, CSAHi) aim to more accurately model and predict arrhythmia risk by including additional human cardiac ion channels in patch clamp electrophysiology screening panels. This broader dataset is then used in sophisticated in silico models of the human ventricular action potential to predict arrhythmias. After 5 years of extensive collaborative effort by working groups in the UK, EU, US and Japan, it has become apparent that incorporating data obtained from the ‘big 6’ panel of cardiac ion channel assays (hERG, Na_V1.5, Ca_V1.2, K_VLQT1, K_V4.3, and K_ir2.1) into standard in silico action potential models allows the accurate prediction of proarrhythmic liability of most, but not all, clinical drugs with well characterised risk profiles.

The voltage-dependent sodium channel, Na_V1.5, was tested on the QPatch in single-hole and multi-hole mode. In this study, we wanted to determine the best test protocol to distinguish between two antiarrhythmic drugs with different modes of action.

The sodium ion channel Na_v1.5 is expressed as in integral membrane protein and contains a tetrodotoxin-resistant voltage-gated sodium channel subunit.The encoded protein is found primarily in cardiac muscle and is responsible for the initial upstroke of the action potential in an electrocardiogram. Mutations in the gene are associated with long QT syndrome type 3, Brugada syndrome, primary cardiac conduction disease and idiopathic ventricular fibrillation.

The slowly activating, delayed rectifier K+ channel is among other activities important for regulating the repolarization phase of cardiac action potentials. The KvLQT1/minK channel consists of two transmembrane proteins. Mutations in the genes KCNQ1/KCNE1 coding for KvLQT1/minK are associated with predisposition to deafness, cardiac arrhythmia syndromes including long QT syndrome, atrial fibrillation and sudden infant death syndrome.

The voltage-gated potassium channel K_V1.5 is a homotetrameric protein present in the heart. It is a delayed rectifier, participating in the early phase of the heart action potential. This report shows data from CHO cells stably expressing K_V1.5 tested on the QPatch platform.

Current clamp recordings provide more physiologically relevant measurements of ion channel activity (in comparison to voltage clamp), allowing the contribution of different ion channel subtypes to resting membrane potential to be evaluated.

Here we demonstrate that:

• Due to their mode of action, some compounds display different
potencies in voltage clamp and current clamp.
• Qube 384 enables drug screening in current clamp mode,
here demonstrated in a cell line stably expressing the KV1.3
ion channel (success rate out of 384 wells of up to 65%).
• Compounds blocking the KV1.3 channel are evaluated for
their ability to depolarize the cell membrane.

Na_V1.7 has moved in the focus of the drug discovery industry in the last years as numerous studies provided strong proof for the efficacy to alleviate both neuropathic and inflammatory pain (Sun, Cohen, & Dehnhardt, 2014). We demonstrate high reproducibility and precision of Na_V1.7 data recorded on Qube

95% of all tested cells had a GΩ seal in the beginning and
61% at the end of a long, 70 min experiment

V_1/2 is stable over time with only a slight shift

Experiments can be performed in physiological ringers,
however fluoride in the internal solution significantly
increases success rate without any altering biophysical
parameters.

CHO-hERG DUO is a QCell, a cell line that can be provided by Sophion. The performance of this particular cell line was evaluated on multi-hole QPlates on QPatch HTX. We have evaluated several features – success rates, stability, pharmacology and biophysical properties of the assay. The report also contains introduction to some of the methodology that is used when running multi-hole experiments on the QPatch.

CHO-hERG DUO is a QCell, a cell line that can be provided by Sophion. The performance of this particular cell line was evaluated on multi-hole QPlates on QPatch HTX. We have evaluated several features – success rates, stability, pharmacology and biophysical properties of the assay. The report also contains introduction to some of the methodology that is used when running multi-hole experiments on the QPatch.

In this report the applications of the CHO TREx-P2X₃ cell line (hereafter called CHO P2X₃) are described. The purpose was to determine the EC₅₀ value for ɑβ-methyl ATP, and the IC₅₀ value of PPADS on the P2X₃ receptors. All data presented were obtained with QPatch and analyzed with the QPatch assay software.

P2X receptors are ligand-gated ion channels (LGIC’s) with time constants of 10-200 ms for activation and 100-200 ms for desensitization (1). The fast kinetics presents a challenge for patch clamp recordings of physiologically realistic P2X receptor currents, and a fast solution exchange technique is required.
This report presents a pharmacological characterization of one of the fastest P2X receptors, the P2X3 receptor, based on perforated patch whole-cell currents from a rat lung epithelial (RLE) cell line stably expressing the human P2X3 receptor. All measurements were recorded with using QPatch.

The P2X3 receptor was targeted with:

-Agonists: CTP (cytidine triphosphate) αβ-me-ATP (α,β-methylene adenosine triphosphate)

-Antagonist: TNP-ATP (trinitrophenyl adenosine triphosphate)

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a chloride channel and controls the regulation of other transport pathways. Mutations in the CFTR gene have been found to cause cystic fibrosis (CF) and congenital bilateral aplasia of the vas deferens (CBAVD).
Here we demonstrate that reliable recordings with the chloride conducting ion channel CFTR can be performed on QPatch using fluoride as activator of adenylate cyclase. We have shown the ability to determine the IC50 for CFTRinh-172 in close correspondence to the literature values.
We can conclude that the overall success in obtaining stable seals and completed experiments are at a level that clearly identifies the CHO–hCFTR assay as feasible and with high quality results on QPatch.

Correct cell handling is important for optimal experimental results. This report gives advice on cell handling in order that you can obtain the best cells possible for use on the QPatch or Qube instruments. Here we describe how to culture your cells and how to reach optimal cell confluence for both cell propagation (“mother flasks”) and for experimental use. Detachment enzymes recommended by Sophion are also described together with methods for harvesting and storing cells.

The voltage-sensitive L-type Ca²⁺-channel (LTCC) Ca_V1.2 is widely expressed in vascular smooth muscle tissue and the heart muscle^1-3. The opening of the channels leads to an increase of intracellular calcium, which acts as second messenger and thereby affects a variety of cellular processes⁴ including heart muscle contraction and neurotransmitter release. Ca_V1.2 is therefore an important target in e.g. safety pharmacology screening. The channels are known to require a large depolarization for their activation and once activated they display a long-lasting current flow, which typically can be blocked by low micromolar concentrations of e.g. dihydropyridines, phenylalkylamines and benzothiazepines^5,6.
In this study, currents from CHO-hCa_V1.2 were recorded on the high-throughput platform Qube 384 in multi-hole mode (10 patch holes per well) at both a customer’s site and at Sophion. Success rates, rundown, sealing properties and the pharmacological effects of two compounds were determined.

The voltage-sensitive L-type Ca²⁺-channel (LTCC) Ca_V1.2 is a crucial component for controlling intracellular activity and thereby essential in the cardiovascular and neuronal system. It is widely expressed in vascular smooth muscle tissue and the heart muscle. The opening of the channels leads to an increase of intracellular calcium, which act as second messenger, and thereby affects a variety of cellular processes including heart muscle contraction and Ca_V1.2 is therefore an important target in e.g. safety pharmacology screening. Ca_V1.2 channels are known to require a large depolarization for their activation and once activated they display a long-lasting current flow, which typically can be blocked by low micromolar concentrations of e.g. dihydropyridines, phenylalkylamines and benzothiazepines. In these studies, currents from HEK-hCa_V1.2 were recorded on the high-throughput platform Qube 384 in both single-hole and multi-hole mode. Success rates, IV characteristics and the pharmacological effects of three different compounds were determined.

The cardiac ion channel encoded by KCNH2, also known as the human ether-à-go-go-related gene (hERG) or KV11.1 can lead to cardiac arrhythmia when blocked and is thus a major off-target in drug discovery. Mutations in this channel can lead to congenital long QT syndrome (LQTS), associated with an irregular heartbeat, fainting and sudden cardiac arrest.
Automated patch clamp (APC) allows assessing the effects of compounds on the hERG channel in the preclinical development process, enabling an early evaluation in drug development. Unattended runs – as we show here on the Qube – are a crucial advantage by saving time and resources. We here report the results of an overnight run with CHO-hERG-Duo (B’SYS) cells at 25˚C, made possible by the Qube supplemented with stacker function and temperature control.