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  • 1.

    【2hr】Expression and Purification of Recombinant Human Apolipoprotein A-II in Pichia pastoris

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    机译 重组人载脂蛋白A-II在毕赤酵母中的表达和纯化
    摘要：Apolipoprotein A-II (ApoA-II) is the second most abundant protein constituent of high-density lipoprotein (HDL). The physiologic role of ApoA-II is poorly defined. ApoA-II may inhibit lecithin:cholesterol acyltransferase and cholesteryl-ester-transfer protein activities, but may increase the hepatic lipase activity. ApoA-II may also inhibit the hepatic cholesteryl uptake from HDL probably through the scavenger receptor class B type I depending pathway. Interpretation of data from transgenic and knockout mice of genes involved in lipoprotein metabolism has been often complicated as clinical implications because of species difference. So it is important to obtain human ApoA-II for further studies about its functions. In our studies, Pichia pastoris expression system was first used to express a high-level secreted recombinant human ApoA-II (rhApoA-II). We have cloned the cDNA encoding human ApoA-II and achieved its high-level secreting expression with a yield of 65 mg/L of yeast culture and the purification process was effective and easy to handle. The purified rhApoA-II can be used to further study its biological activities.
  • 2.

    【2hr】Development of Multiple Cell-Based Assays for the Detection of Histone H3 Lys27 Trimethylation (H3K27me3)

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    机译 用于检测组蛋白H3 Lys27三甲基化（H3K27me3）的多种基于细胞的检测方法的开发
    摘要：Posttranslational modification of histone proteins in eukaryotes plays an important role in gene transcription and chromatin structure. Dysregulation of the enzymes involved in histone modification has been linked to many cancer forms, making this target class a potential new area for therapeutics. A reliable assay to monitor small-molecule inhibition of various epigenetic enzymes should play a critical role in drug discovery to fight cancer. However, it has been challenging to develop cell-based assays for high-throughput screening (HTS) and compound profiling. Recently, two homogeneous cell-based assay kits using the AlphaLISA® and LanthaScreen® technologies to detect trimethyl histone H3 Lysine 27 have become commercially available, and a heterogeneous cell assay with modified dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA®) format has been reported. To compare their pros and cons, we evaluated, optimized, and validated these three assay formats in three different cell lines and compared their activities with traditional Western blot detection of histone methylation inhibition by using commercial and in-house small-molecule inhibitors. Our data indicate that, although all four formats produced acceptable results, the homogeneous AlphaLISA assay was best suited for HTS and compound profiling due to its wider window and ease of automation. The DELFIA and Western blot assays were useful as validation tools to confirm the cell activities and eliminate potential false-positive compounds.
  • 3.

    【2hr】Measuring Interference of Drug-Like Molecules with the Respiratory Chain: Toward the Early Identification of Mitochondrial Uncouplers in Lead Finding

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    机译 测量药物分子与呼吸链的干扰：早期发现铅中的线粒体解偶联剂。
    摘要：The electron transport chain (ETC) couples electron transfer between donors and acceptors with proton transport across the inner mitochondrial membrane. The resulting electrochemical proton gradient is used to generate chemical energy in the form of adenosine triphosphate (ATP). Proton transfer is based on the activity of complex I–V proteins in the ETC. The overall electrical activity of these proteins can be measured by proton transfer using Solid Supported Membrane technology. We tested the activity of complexes I, III, and V in a combined assay, called oxidative phosphorylation assay (oxphos assay), by activating each complex with the corresponding substrate. The oxphos assay was used to test in-house substances from different projects and several drugs currently available on the market that have reported effects on mitochondrial functions. The resulting data were compared to the influence of the respective compounds on mitochondria as determined by oxygen consumption and to data generated with an ATP depletion assay. The comparison shows that the oxidative phosphorylation assay provides both a rapid approach for detecting interaction of compounds with respiratory chain proteins and information on their mode of interaction. Therefore, the oxphos assay is a useful tool to support structure activity relationship studies by allowing early identification of mitotoxicity and for analyzing the outcome of phenotypic screens that are susceptible to the generation of mitotoxicity-related artifacts.
  • 4.

    【2hr】A Highly Specific Cell-Based High-Throughput Screening Assay for Ligands of Cyclic Adenosine Monophosphate Receptor Protein in Gram-Negative Bacteria

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    机译 革兰氏阴性细菌中环腺苷单磷酸受体蛋白配体的高度特异性基于细胞的高通量筛选方法
    摘要：Quorum sensing is a cell–cell communication process in bacteria that involves the production, release, and subsequent detection of chemical signal molecules called autoinducers. In Vibrio cholerae, multiple input signals activate the expression of the quorum sensing regulator HapR, which acts to repress the expression of virulence factors. We have shown that CRP, the cyclic adenosine monophosphate (cAMP) receptor protein, enhances quorum sensing by activating the biosynthesis of cholera autoinducer 1, the major signaling molecule that contributes to the activation of HapR. Thus, proquorum sensing CRP agonists could inhibit virulence and lead to new drugs to treat severe cholera. In this study, we show that expression of the quorum sensing-regulated luxCDABE operon can be used as a robust readout for CRP activity. Further, we describe and validate a highly specific cell-based luminescence high-throughput screening assay for proquorum sensing CRP ligands. A pilot screen of 9,425 compounds yielded a hit rate of 0.02%, one hit being cAMP itself. The Z′ value for this assay was 0.76 and its coefficient of variance 8% for the positive control compound. To our knowledge, this is the first cell-based assay for ligands of the highly conserved CRP protein of Gram-negative bacteria. The use of this assay to screen large chemical libraries could identify lead compounds to treat cholera, as well as small molecules to probe ligand–receptor interactions in the CRP molecule.
  • 5.

    【2hr】A Time-Resolved Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of 14-3-3 Protein–Protein Interaction Inhibitors

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    机译 高分辨筛选14-3-3蛋白质-蛋白质相互作用抑制剂的时间分辨荧光共振能量转移测定
    摘要：Protein–protein interaction networks mediate diverse biological processes by regulating various signaling hubs and clusters. 14-3-3 proteins, a family of phosphoserine/threonine-binding molecules, serve as major interaction hubs in eukaryotic cells and have emerged as promising therapeutic targets for various human diseases. In order to identify chemical probes for mechanistic studies and for potential therapeutic development, we have developed highly sensitive bioassays to monitor the interaction of 14-3-3 with a client protein. In this study, we describe a homogenous time-resolved fluorescence resonance energy transfer (TR-FRET) assay to detect the interaction of 14-3-3 with Bad, a proapoptotic member of the Bcl-2 family. Through a series of titration studies in which europium-labeled 14-3-3 serves as an FRET donor and a Dy647-labeled phosphorylated Bad, the peptide acts as an FRET acceptor, we have achieved a robust TR-FRET assay that is suitable for high-throughput screening (HTS) with an excellent signal-to-background ratio of >20 and Z′ values >0.7. This assay was further miniaturized to a 1,536-well format for ultra-HTS (uHTS), and exhibited a similar robust performance. The utility and performance of the assay for uHTS were validated by (i) known inhibitors, including peptide R18 and small molecule FOBISIN101, and (ii) screening of a 51,200 compound library. This simple and robust assay is generally applicable to detect the interaction of 14-3-3 with other client proteins. It provides a sensitive and easy-to-use tool to facilitate the discovery of 14-3-3 protein inhibitors as well as to study 14-3-3-mediated protein–protein interactions.
  • 6.

    【2hr】Interview with John W. Kozarich, PhD

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    机译 John W. Kozarich博士访谈
    • 作者：Vicki Glaser
    • 刊名：Assay and Drug Development Technologies
    • 2013年第5期
    摘要：
  • 7.

    【2hr】Assay Development for Histone Methyltransferases

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    机译 组蛋白甲基转移酶的测定开发
    摘要：Epigenetic modifications play a crucial role in human diseases. Unlike genetic mutations, however, they do not change the underlying DNA sequences. Epigenetic phenomena have gained increased attention in the field of cancer research, with many studies indicating that they are significantly involved in tumor establishment and progression. Histone methyltransferases (HMTs) are a large group of enzymes that specifically methylate protein lysine and arginine residues, especially in histones, using S-adenosyl-l-methionine (SAM) as the methyl donor. However, in general, HMTs have no widely accepted high-throughput screening (HTS) assay format, and reference inhibitors are not available for many of the enzymes. In this study, we describe the application of a miniaturized, radioisotope-based reaction system: the HotSpotSM platform for methyltransferases. Since this platform employs tritiated SAM as a cofactor, it can be applied to the assay of any HMT. The key advantage of this format is that any substrate can be used, including peptides, proteins, or even nucleosomes, without the need for labeling or any other modifications. Using this platform, we have determined substrate specificities, characterized enzyme kinetics, performed compound profiling for both lysine and arginine methyltransferases, and carried out HTS for a small-library LOPAC against DOT1L. After hit confirmation and profiling, we found that suramin inhibited DOT1L, NSD2, and PRMT4 with IC50 values at a low μM range.
  • 8.

    【2hr】The Tenth Annual Ion Channel Retreat, Vancouver, Canada, June 25–27, 2012

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    机译 第十届年度离子通道务虚会，加拿大温哥华，2012年6月25日至27日
    摘要：Ten years after Aurora Biomed (Vancouver, British Columbia, Canada) hosted the inaugural Ion Channel Retreat, this event is recognized as a leading conference for ion channel researchers. Held annually in Vancouver, this meeting consistently provides an outlet for researchers to share their findings while learning about new concepts, methods, and technologies. Researchers use this forum to discuss and debate a spectrum of topics from ion channel research and technology to drug discovery and safety. The Retreat covered key subjects in the ion channel industry, including ion channels as disease targets, transient receptor protein channels as pain and disease targets, ion channels as pain targets, ion channel structure and function, ion channel screening technologies, cardiac safety and toxicology, and cardiac function and pharmacology.
  • 9.

    【2hr】Label-Free Cytotoxicity Screening Assay by Digital Holographic Microscopy

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    机译 数字全息显微镜的无标记细胞毒性筛选测定
    摘要：We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z′-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose–response curves and known IC50 values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.
  • 10.

    【2hr】Development and Validation of a High-Content Screening Assay to Identify Inhibitors of Cytoplasmic Dynein-Mediated Transport of Glucocorticoid Receptor to the Nucleus

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    机译 开发和验证的高内涵筛选测定法，以鉴定胞质动力蛋白介导的糖皮质激素受体向核转运的抑制剂。
    摘要：Rapid ligand-induced trafficking of glucocorticoid nuclear hormone receptor (GR) from the cytoplasm to the nucleus is an extensively studied model for intracellular retrograde cargo transport employed in constructive morphogenesis and many other cellular functions. Unfortunately, potent and selective small-molecule disruptors of this process are lacking, which has restricted pharmacological investigations. We describe here the development and validation of a 384-well high-content screening (HCS) assay to identify inhibitors of the rapid ligand-induced retrograde translocation of cytoplasmic glucocorticoid nuclear hormone receptor green fluorescent fusion protein (GR-GFP) into the nuclei of 3617.4 mouse mammary adenocarcinoma cells. We selected 3617.4 cells, because they express GR-GFP under the control of a tetracycline (Tet)-repressible promoter and are exceptionally amenable to image acquisition and analysis procedures. Initially, we investigated the time-dependent expression of GR-GFP in 3617.4 cells under Tet-on and Tet-off control to determine the optimal conditions to measure dexamethasone (Dex)-induced GR-GFP nuclear translocation on the ArrayScan-VTI automated imaging platform. We then miniaturized the assay into a 384-well format and validated the performance of the GR-GFP nuclear translocation HCS assay in our 3-day assay signal window and dimethylsulfoxide validation tests. The molecular chaperone heat shock protein 90 (Hsp90) plays an essential role in the regulation of GR steroid binding affinity and ligand-induced retrograde trafficking to the nucleus. We verified that the GR-GFP HCS assay captured the concentration-dependent inhibition of GR-GFP nuclear translocation by 17-AAG, a benzoquinone ansamycin that selectively blocks the binding and hydrolysis of ATP by Hsp90. We screened the 1280 compound library of pharmacologically active compounds set in the Dex-induced GR-GFP nuclear translocation assay and used the multi-parameter HCS data to eliminate cytotoxic compounds and fluorescent outliers. We identified five qualified hits that inhibited the rapid retrograde trafficking of GR-GFP in a concentration-dependent manner: Bay 11-7085, 4-phenyl-3-furoxancarbonitrile, parthenolide, apomorphine, and 6-nitroso-1,2-benzopyrone. The data presented here demonstrate that the GR-GFP HCS assay provides an effective phenotypic screen and support the proposition that screening a larger library of diversity compounds will yield novel small-molecule probes that will enable the further exploration of intracellular retrograde transport of cargo along microtubules, a process which is essential to the morphogenesis and function of all cells.
  • 11.

    【2hr】Preparation and Activity Analysis of Recombinant Human High-Density Lipoprotein

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    机译 重组人高密度脂蛋白的制备及活性分析
    摘要：Population studies have consistently shown a highly inverse correlation between plasma concentration of high-density lipoprotein and the risk of atherosclerotic cardiovascular disease in humans. High-density lipoprotein (HDL) as a therapeutic target is an intense area of ongoing investigation. Aiming to solve the shortcomings of native HDL application, we prepared recombinant human HDL (rhHDL) that contains a similar composition and has similar functions with native HDL. Six kinds of recombinant human apolipoproteins (rhapo)—rhapoA-I, rhapoA-II, rhapoA-IV, rhapoC-I, rhapoC-II, and rhapoE—were expressed in Pichia pastoris and purified with chromatography. By the facilitation of cholate, six kinds of rhapo penetrated among the phosphatidylcholine acyl chains. After purification by density-gradient centrifugation, rhHDL was acquired. Based on morphological observation, we confirmed that the micellar complexes of rhapo with phosphatidylcholine and cholesterol were prepared. We carried on comparative studies in vitro and in vivo between native HDL and rhHDL. Cellular cholesterol efflux assays showed that rhHDL could promote the efflux of excess cholesterol from macrophages. Furthermore, rhHDL has similar effects with native HDL on the blood lipid metabolism in hyperlipidemic mice. In conclusion, rhHDL has similar effects on antiatherosclerosis with native HDL through reverse cholesterol transport, antioxidative, and antithrombotic properties. It could be used as a therapeutic HDL-replacement agent.
  • 12.

    【2hr】The XTT Cell Proliferation Assay Applied to Cell Layers Embedded in Three-Dimensional Matrix

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    机译 XTT细胞增殖测定法应用于嵌入三维矩阵的细胞层
    摘要：Cell proliferation, a main target in cancer therapy, is influenced by the surrounding three-dimensional (3D) extracellular matrix (ECM). In vitro drug screening is, thus, optimally performed under conditions in which cells are grown (embedded or trapped) in dense 3D matrices, as these most closely mimic the adhesive and mechanical properties of natural ECM. Measuring cell proliferation under these conditions is, however, technically more challenging compared with two-dimensional (2D) culture and other “3D culture conditions,” such as growth on top of a matrix (pseudo-3D) or in spongy scaffolds with large pore sizes. Consequently, such measurements are only slowly applied on a wider scale. To advance this, we report on the equal quality (dynamic range, background, linearity) of measuring the proliferation of cell layers embedded in dense 3D matrices (collagen, Matrigel) compared with cells in 2D culture using the easy (one-step) and in 2D well-validated, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)-assay. The comparison stresses the differences in proliferation kinetics and drug sensitivity of matrix-embedded cells versus 2D culture. Using the specific cell-layer-embedded 3D matrix setup, quantitative measurements of cell proliferation and cell invasion are shown to be possible in similar assay conditions, and cytostatic, cytotoxic, and anti-invasive drug effects can thus be reliably determined and compared in physiologically relevant settings. This approach in the 3D matrix holds promise for improving early-stage, high-throughput drug screening, targeting either highly invasive or highly proliferative subpopulations of cancers or both.
  • 13.

    【2hr】Simultaneous Measurement of 10,000 Protein-Ligand Affinity Constants Using Microarray-Based Kinetic Constant Assays

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    机译 使用基于微阵列的动力学常数测定法同时测量10,000个蛋白质-配体亲和力常数
    摘要：Fluorescence-based endpoint detection of microarrays with 10,000 or more molecular targets is a most useful tool for high-throughput profiling of biomolecular interactions, including screening large molecular libraries for novel protein ligands. However, endpoint fluorescence data such as images of reacted microarrays contain little information on kinetic rate constants, and the reliability of endpoint data as measures of binding affinity depends on reaction conditions and postreaction processing. We here report a simultaneous measurement of binding curves of a protein probe with 10,000 molecular targets in a microarray with an ellipsometry-based (label-free) optical scanner. The reaction rate constants extracted from these curves (kon, koff, and ka=kon/koff) are used to characterize the probe-target interactions instead of the endpoints. This work advances the microarray technology to a new milestone, namely, from an endpoint assay to a kinetic constant assay platform. The throughput of this binding curve assay platform is comparable to those at the National Institutes of Health Molecular Library Screening Centers, making it a practical method in screening compound libraries for novel ligands and for system-wide affinity profiling of proteins, viruses, or whole cells against diverse molecular targets.
  • 14.

    【2hr】A Framework for Image-Based Classification of Mitotic Cells in Asynchronous Populations

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    机译 基于图像的异步群体中有丝分裂细胞分类的框架。
    摘要：High content screening (HCS) has emerged an important tool for drug discovery because it combines rich readouts of cellular responses in a single experiment. Inclusion of cell cycle analysis into HCS is essential to identify clinically suitable anticancer drugs that disrupt the aberrant mitotic activity of cells. One challenge for integration of cell cycle analysis into HCS is that cells must be chemically synchronized to specific phases, adding experimental complexity to high content screens. To address this issue, we have developed a rules-based method that utilizes mitotic phosphoprotein monoclonal 2 (MPM-2) marker and works consistently in different experimental conditions and in asynchronous populations. Further, the performance of the rules-based method is comparable to established machine learning approaches for classifying cell cycle data, indicating the robustness of the features we use in the framework. As such, we suggest the use of MPM-2 analysis and its associated expressive features for integration into HCS approaches.
  • 15.

    【2hr】Kinetic Measurements Using Flow Cytometry: New Methods for Monitoring Intracellular Processes

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    机译 使用流式细胞仪进行动力学测量：监测细胞内过程的新方法
    摘要：The aim of our work was to establish flow cytometry methods for the characterization of mitochondrial Ca2+ levels, plasma membrane potential, and superoxide generation and to relate kinetics to that of cytoplasmic Ca2+ levels during short-term activation of T-lymphocytes. We monitored the change of fluorescence absorbance of sequentially measured Jurkat cells for 12 min. The cells were stained with the fluorescent dyes Fluo3-AM, Rhod2/AM, di-BA-C4-(5), or dihydroethidium and then were stimulated with increasing doses of phytohemagglutinin (PHA) or were treated with rotenone. Double-logistic function was fitted to cytoplasmic Ca2+ signal and mitochondrial Ca2+ levels, whereas logistic function was fitted to plasma membrane potential and superoxide levels. The calculated function parameters were area under the curve (AUC), maximum (Max), time to reach maximum (tmax), slope at the first 50% value of Max (Slope), and ending (End) values, respectively. We found significant dose–response relationship between PHA dose and cytoplasmic Ca2+ signals (AUC, Max, Slope: P<0.05), mitochondrial Ca2+ levels (AUC and Max: P<0.05), and plasma membrane potential (AUC and End values: P<0.05). In rotenone-treated cells, superoxide generation increased in a dose-dependent manner (P<0.05 for AUC and End values, respectively). The present methodology provides an opportunity for monitoring and characterizing mitochondrial Ca2+ levels, plasma membrane potential, and superoxide generation in PHA-activated or rotenone-treated Jurkat cells with flow cytometry.
  • 16.

    【2hr】Dynamic Mass Redistribution Assays Decode Surface Influence on Signaling of Endogenous Purinergic P2Y Receptors

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    机译 动态质量重分布分析可解码表面对内源性嘌呤能P2Y受体信号传导的影响
    摘要：Interactions with extracellular matrices (ECM) shape the signaling and functions of many types of cells and receptors, and distinct ECM coatings have been used in a wide range of substrates for drug discovery processes. Here, we investigate the influence of ECM protein coatings on the signaling of endogenous purinergic 2Y (P2Y) receptors in human embryonic kidney HEK293 cells using dynamic mass redistribution (DMR) assays enabled by label-free optical biosensor. Results showed that ECM proteins had significant impacts on the DMR characteristics, potency, and efficacy of seven P2Y agonists. This study documents the importance of surface chemistry in regulating receptor signaling.
  • 17.

    【2hr】Assessment of Compound Hepatotoxicity Using Human Plateable Cryopreserved Hepatocytes in a 1536-Well-Plate Format

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    机译 使用1536孔板格式的人类可平板冷冻保存的肝细胞评估化合物的肝毒性
    摘要：Hepatotoxicity is a major concern for both drug development and toxicological evaluation of environmental chemicals. The assessment of compound-induced hepatotoxicity has traditionally relied on in vivo testing; however, it is being replaced by human in vitro models due to an emphasis on the reduction of animal testing and species-specific differences. Since most cell lines and hybridomas lack the full complement of enzymes at physiological levels found in the liver, primary hepatocytes are the gold standard to study liver toxicities in vitro due to the retention of most of their in vivo activities. Here, we optimized a cell viability assay using plateable cryopreserved human hepatocytes in a 1536-well-plate format. The assay was validated by deriving inhibitory concentration at 50% values for 12 known compounds, including tamoxifen, staurosporine, and phenylmercuric acetate, with regard to hepatotoxicity and general cytotoxicity using multiple hepatocyte donors. The assay performed well, and the cytotoxicity of these compounds was confirmed in comparison to HepG2 cells. This is the first study to report the reliability of using plateable cryopreserved human hepatocytes for cytotoxicity studies in a 1536-well-plate format. These results suggest that plateable cryopreserved human hepatocytes can be scaled up for screening a large compound library and may be amenable to other hepatocytic assays such as metabolic or drug safety studies.
  • 18.

    【2hr】A Suite of Modular Fluorescence Assays Interrogate the Human Immunodeficiency Virus Glycoprotein-41 Coiled Coil and Assist in Determining Binding Mechanism of Low Molecular Weight Fusion Inhibitors

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    机译 一套模块化的荧光测定法审问人类免疫缺陷病毒糖蛋白41螺旋线圈，并协助确定低分子量融合抑制剂的结合机制。
    • 作者：Miriam Gochin
    • 刊名：Assay and Drug Development Technologies
    • 2012年第5期
    摘要：Several different segments of the gp41 N-heptad repeat coiled coil have been constructed using N-terminal bipyridyl modification of composite peptides and inducing trimerization by adding ferrous ions. These metallopeptides act as receptors in fluorescence-binding assays with corresponding fluorescently labeled C-peptide probes. The FeII coordination complex quenches C-peptide fluorescence upon binding, and reversal of quenching by a small molecule inhibitor can be used to obtain the inhibitor-binding constant. A total of 10 peptide pairs targeting 25–46 residue segments of the coiled coil were constructed, with C-peptide probes of different lengths and binding affinities. The result is a suite of assays for exploring binding in the mM to nM range to any desired region of the coiled coil, including the hydrophobic pocket (HP), extended regions on either side of the pocket, or a region associated with T20 resistance mutations. These assays are high-throughput ready, and could be used to discover novel compounds binding along various regions of the gp41 coiled coil groove. They were used to evaluate a sub-μM low molecular weight fusion inhibitor, resulting in the finding that the molecule bound specifically to the HP and attained its potency from a low off-rate.
  • 19.

    【2hr】A Cell-Based Assay for Screening Spindle Checkpoint Inhibitors

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    机译 筛选主轴检查点抑制剂的基于细胞的分析
    摘要：In eukaryotes, the spindle checkpoint acts as a surveillance mechanism that ensures faithful chromosome segregation. The spindle checkpoint prevents premature separation of sister chromatids and the onset of anaphase until every chromosome is properly attached to the mitotic spindle. Tumorigenesis might result from generation of aneuploidy by dysfunction of the spindle checkpoint. Differences of the checkpoint system in normal cells versus tumor cells might provide a new opportunity in cancer drug development; therefore, efforts to identify the spindle checkpoint inhibitors have been fostered. Based on spindle checkpoint inhibitors being able to induce cells to exit mitotic arrest caused by microtubule drug treatment, we developed a cell-based assay to screen compounds that were potential spindle checkpoint inhibitors. This assay was validated with a known spindle checkpoint inhibitor and was easy to adapt to a large-scale screening. It also had the advantages of being high in sensitivity and low in cost.
  • 20.

    【2hr】Comparison of Cell Expression Formats for the Characterization of GABAA Channels Using a Microfluidic Patch Clamp System

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    机译 使用微流体膜片钳系统比较GABAA通道的细胞表达格式
    摘要：Ensemble recording and microfluidic perfusion are recently introduced techniques aimed at removing the laborious nature and low recording success rates of manual patch clamp. Here, we present assay characteristics for these features integrated into one automated electrophysiology platform as applied to the study of GABAA channels. A variety of cell types and methods of GABAA channel expression were successfully studied (defined as IGABA>500 pA), including stably transfected human embryonic kidney (HEK) cells expressing α1β3γ2 GABAA channels, frozen ready-to-assay (RTA) HEK cells expressing α1β3γ2 or α3β3γ2 GABAA channels, transiently transfected HEK293T cells expressing α1β3γ2 GABAA channels, and immortalized cultures of human airway smooth muscle cells endogenously expressing GABAA channels. Current measurements were successfully studied in multiple cell types with multiple modes of channel expression in response to several classic GABAA channel agonists, antagonists, and allosteric modulators. We obtained success rates above 95% for transiently or stably transfected HEK cells and frozen RTA HEK cells expressing GABAA channels. Tissue-derived immortalized cultures of airway smooth muscle cells exhibited a slightly lower recording success rate of 75% using automated patch, which was much higher than the 5% success rate using manual patch clamp technique by the same research group. Responses to agonists, antagonists, and allosteric modulators compared well to previously reported manual patch results. The data demonstrate that both the biophysics and pharmacologic characterization of GABAA channels in a wide variety of cell formats can be performed using this automated patch clamp system.
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