Miniaturization of whole live cell-based GPCR assays using microdispensing and detection systems

Cell-based beta-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 microL total assay volume in a 3456-well microplate. Studies were done to evaluate both receptor agonists and antagonists. The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC(50)/IC(50) precision. These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h. The instrumentation used and practical aspects of the assay development are discussed.     

Evaluation of division-arrested cells for cell-based high-throughput screening and profiling

Just-in-time cell supply for cell-based high-throughput screening (HTS) is frequently problematic. In addition to scheduling and logistical issues, quality issues and variability due to passage effect, cell cycle, or confluency contribute to day-to-day signal variability in the course of cell-based HTS campaigns. Cell division-arrest and cryopreservation technologies permit the use of cells as assay-ready reagents for HTS and other cell-based profiling and structure-activity studies. In this report, the authors compare division-arrested and dividing cells in 2 assay types that are dependent on movement of proteins within or through cell membranes: a receptor tyrosine kinase assay involving A431 cells responsive to epidermal growth factor, and a secretion reporter assay, which measures secretion of a reporter gene, secreted alkaline phosphatase. In both assays, dividing and division-arrested cells yielded similar basal and maximal signals at a given cell density. Similar IC50s were obtained for reference inhibitors in each assay, type in both dividing and division-arrested cells. In addition, for the secretion reporter assay, when comparing IC50s obtained from 44 compounds randomly chosen from a primary screening hit list, the rank order of potency obtained from dividing cells and division-arrested cells was essentially identical. Furthermore, the results show that, under certain assay conditions, data generated using division-arrested cells are less variable than those generated using dividing cells. In summary, the results suggest that, in many cases, division-arrested cells can substitute for dividing cells and offer certain advantages for cell-based assays.     

Cell lysis with dimethyl sulphoxide produces stable homogeneous solutions in the dichlorofluorescein oxidative stress assay

The oxidation of 2',7'-dichlorodihydrofluorescein (2',7'-dichlorofluorescin, DCFH) to a fluorescent product, 2',7'-dichlorofluorescein (DCF), is commonly used to quantitatively measure oxidative stress in cells using a fluorescence microplate reader. However, many cell lines tend to grow non-uniformly in the wells. This non-uniform distribution results in a high degree of variability in the fluorescence signal and decreases the precision of the method. Also, samples treated in large culture plates, dishes or flasks cannot be assayed directly in fluorescence microplate readers. This study reports an improved DCF assay method that lyses cells with DMSO/PBS (90% dimethyl sulphoxide/10% phosphate buffered saline). Oxidative stress was induced with either hydrogen peroxide or an hypoxia-reoxygenation treatment. Cell lysis with DMSO/PBS resulted in highly stable fluorescence signals in comparison to Triton X-100/PBS lysed cells. The precision of DCF fluorescence measurements of DMSO/PBS lysed cells was much better than for attached cells measured directly in 96-well plates. While DCF fluorescence in PBS was strongly quenched by albumin, no quenching occurred in DMSO/PBS. In conclusion this study describes a more convenient and accurate method for measuring cellular oxidative stress that also makes it possible to assay cells treated in large culture plates.     

Cell lysis with dimethyl sulphoxide produces stable homogeneous solutions in the dichlorofluorescein oxidative stress assay

The oxidation of 2′,7′-dichlorodihydrofluorescein (2′,7′-dichlorofluorescin, DCFH) to a fluorescent product, 2′,7′-dichlorofluorescein (DCF), is commonly used to quantitatively measure oxidative stress in cells using a fluorescence microplate reader. However, many cell lines tend to grow non-uniformly in the wells. This non-uniform distribution results in a high degree of variability in the fluorescence signal and decreases the precision of the method. Also, samples treated in large culture plates, dishes or flasks cannot be assayed directly in fluorescence microplate readers. This study reports an improved DCF assay method that lyses cells with DMSO/PBS (90% dimethyl sulphoxide/10% phosphate buffered saline). Oxidative stress was induced with either hydrogen peroxide or an hypoxia-reoxygenation treatment. Cell lysis with DMSO/PBS resulted in highly stable fluorescence signals in comparison to Triton X-100/PBS lysed cells. The precision of DCF fluorescence measurements of DMSO/PBS lysed cells was much better than for attached cells measured directly in 96-well plates. While DCF fluorescence in PBS was strongly quenched by albumin, no quenching occurred in DMSO/PBS. In conclusion this study describes a more convenient and accurate method for measuring cellular oxidative stress that also makes it possible to assay cells treated in large culture plates.     

Miniaturization of a Mammalian Cell-Based Assay: Luciferase Reporter Gene Readout in a 3 Microliter 1536-Well Plate

The combined efforts of the fields of combinatorial chemistry and genomics have significantly increased the number of compounds and therapeutic targets available for screening. The number of compounds will reach into the million range in the near future and provide vast chemical diversity for drug discovery. However, this reservoir of chemical diversity creates downstream hurdles for any screening effort. Properly examining this number of compounds increases investments dramatically, both in the number of dollars spent and amount of limited reagents depleted. Traditional HTS techniques, such as the use of 96-well microtiter plates, have paved the way for faster processing speeds, but are being rapidly overwhelmed by screening demands. Miniaturization of such assays will allow for greater throughput, while concurrently reducing cost. To date, miniaturization efforts have been most successfully applied to bacterial and soluble protein based assays. Questions about the ability to deliver microquantities of mammalian cells without disruption of the cell membrane and/or activation of stress responses have been raised. An assay has been developed in which a human T-cell screen has been adapted to a 1536-well plate format. Through the use of a luciferase reporter gene system, it is shown that a mammalian cell-based assay may be successfully performed in 3 μl and potent inhibitors of the target of interest identified.     

Cell-based bioluminescent assays for all three proteasome activities in a homogeneous format

A luminescent method to individually measure the chymotrypsin-like, trypsin-like, or caspase-like activities of the proteasome in cultured cells was developed. Each assay uses a specific luminogenic peptide substrate in a buffer optimized for cell permeabilization, proteasome activity, and luciferase activity. Luminescence is generated in a coupled-enzyme format in which proteasome cleavage of the peptide conjugated substrate generates aminoluciferin, which is a substrate for luciferase. The homogeneous method eliminates the need to prepare individual cell extracts as samples. Luminogenic proteasome substrates and buffer formulations enabled development of a single reagent addition method with adequate sensitivity for 96- and 384-well plate formats. Proteasome trypsin-like specificity was enhanced by incorporating a mixture of protease inhibitors that significantly reduce nonspecific serum and cellular backgrounds. The assays were used to determine EC₅₀ values for the specific proteasome inhibitors epoxomicin and bortezomib for each of the catalytic sites using a variety of cancer lines. These cell-based proteasome assays are direct, simple, and sensitive, making them ideal for high-throughput screening.     

Miniaturization and validation of a sensitive multiparametric cell-based assay for the concomitant detection of microtubule-destabilizing and microtubule-stabilizing agents

The authors describe a cell-based assay for anti-microtubule compounds suitable for automation. This assay allows the identification, in a single screening campaign, of both microtubule-destabilizing and microtubule-stabilizing agents. Its rationale is based on the substrate properties of the tubulin-modifying enzymes involved in the tubulin tyrosination cycle. This cycle involves the removal of the C-terminal tyrosine of the tubulin alpha-subunit by an ill-defined tubulin carboxypeptidase and its readdition by tubulin tyrosine ligase. Because of the substrate properties of these enzymes, dynamic microtubules, sensitive to depolymerizing drugs, are composed of tyrosinated tubulin, whereas non-dynamic, stabilized microtubules are composed of detyrosinated tubulin. Thus depolymerization or stabilization of the microtubule network can easily be detected with double-immunofluorescence staining using antibodies specific to tyrosinated and detyrosinated tubulin. The authors have scaled this assay to the 96-well plate format and adapted its process for an automated handling, including a readout using a microplate reader. They describe the different steps of this adaptation. This assay was validated using known compounds. This new cell-based assay represents an alternative to both global cytotoxicity assays and in vitro tubulin assembly assays commonly used for the detection of microtubule poisons.     

T-cell line for HIV drug screening using EGFP as a quantitative marker of HIV-1 replication

The rapid increase of viral strains that are resistant to the currently available antiretroviral drugs is a threat to the success of current human immunodeficiency virus type 1 (HIV-1) treatment and emphasizes the importance of developing novel anti-HIV-1 compounds. To improve the current abilities to screen for novel HIV-1 inhibitors, here we introduce a T-cell-based reporter cell line (JLTRG-RS) that expresses both HIV-1 coreceptors, CXCR4 and CCRS, and provides the convenience of using enhanced green fluorescent protein (EGFP) as a direct and quantitative marker. Unlike previous EGFP-based reporter cell lines, JLTRG-RS cells have an unusually high dynamic signal range, sufficient for plate reader detection using a 384-well format. In this format, JLTRG-R5 cell-based infectivity assays have a Z'-factor of 0.78, which defines the assay as extremely robust and clearly amenable to high-throughput screening. The functional similarity of the JLTRG-R5 cell line and peripheral blood mononuclear cells (PBMCs) was demonstrated through the identity of the inhibitory concentrations, 50% (IC50s) for four antiretroviral compounds or neutralizing antibodies. Because EGFP can be directly and continuously quantified in cell culture, the reporter cell line requires no manipulation during assay preparation or analysis. In addition, the EGFP marker allows for data acquisition at an optimal time point by prescreening selected positive control wells using fluorescent microscopy. These characteristics make the system extremely flexible, rapid, and inexpensive. Due to its intrinsic flexibility, the JLTRG-R5 cell-based reporter system provides a powerful tool to greatly facilitate future screening for HIV-1 inhibitors.     

A single secreted luciferase-based gene reporter assay

Promoter analysis typically employs a reporter gene fused to a test promoter combined with a second reporter fused to a control promoter that is used for normalization purposes. However, this approach is not valid when experimental conditions affect the control promoter. We have developed and validated a single secreted luciferase reporter (SSLR) assay for promoter analysis that avoids the use of a control reporter. The approach uses an early level of expression of a secreted luciferase linked to a test promoter as an internal normalization control for subsequent analysis of the same promoter. Comparison of the SSLR assay with the dual luciferase reporter (DLR) assay using HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) and LDLR (low-density lipoprotein receptor) promoter constructs, which are down-regulated by 25-hydroxycholesterol, show that both assays yield similar results. Comparison of the response of the HMGCR promoter in SSLR transient assays compared very favorably with the response of the same promoter in the stable cell line. Overall, the SSLR assay proved to be a valid alternative to the DLR assay for certain applications and had significant advantages in that measurement of only one luciferase is required and monitoring can be continuous because cell lysis is not necessary.     

Automated triplexed hepatocyte-based viability and CYP1A and -3A induction assays

Cytochrome P450 (CYP) enzymes are key players in drug metabolism. Therefore, it is essential to understand how these enzymes can be affected by xenobiotics with regards to induction and toxicity to avoid potential drug-drug interactions. Typically, information has been gathered by combining data from multiple experiments, which is time-consuming and labor intensive, and interassay variability may lead to misinterpretation. Monitoring CYP induction and cytotoxicity by xenobiotics using an automated, multiplexed format can decrease workload and increase data confidence. Here the authors demonstrate the ability to monitor CYP1A and CYP3A4 induction, combined with a cytotoxicity measurement, from a single microplate well using cryopreserved human hepatocytes. The assay procedure was automated in a 384-well format, including cell manipulations, compound titration and transfer, and reagent dispensing, using simple robotic instrumentation. EC(50) and E(max) values were derived for multiple known CYP1A and -3A4 inducers. Induction and toxicological responses in the triplex system were validated based on literature values from conventional single-parameter assays. Validation and pharmacology data confirm that multiplexed cell-based CYP assays can simplify workload, save time and effort, and generate biologically relevant data.     

Stable luciferase reporter cell lines for signal transduction pathway readout using GAL4 fusion transactivators

While GAL4 fusion activators have been widely used for dissecting signal transduction pathways in transient assays, there has been surprisingly little reported on utilizing cell lines with stably integrated fusion activators. To avoid problems with the efficiency and reproducibility inherent to transient transfection, we describe here the generation and characterization of HeLa reporter cell lines, which contain a stably integrated luciferase gene responsive to stably integrated and constitutively expressed GAL4-CREB or GAL4-Elk1 fusion activators. These cell lines exhibited extremely low basal luciferase expression but robust response to various extracellular stimuli or the expression of signaling molecules that resulted in elevated MAP kinase or PKA activities. This integrated two-component reporter system allows one to focus specifically on particular signaling pathway endpoints and the altered transactivation activity of either Elk1 or CREB. With the procedures described here, many novel cell-based assays can be developed by generating new reporter cell lines with medically important but difficult-to-transfect cell types, and by using different reporter genes or different fusion transactivator genes.     

Optimization procedure for small interfering RNA transfection in a 384-well format

High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format. To overcome these difficulties, the authors developed an approach using a viral-based transfection system that measures siRNA efficacy in a standardized 384-well assay. This method allows measurement of siRNA activity in a phenotypically neutral manner by quantifying the knockdown of an exogenous luciferase gene delivered by a lentiviral vector. In this assay, the efficacy of a luciferase siRNA is compared to a negative control siRNA across many distinct assay parameters including cell type, cell number, lipid type, lipid volume, time of the assay, and concentration of siRNA. Once the siRNA transfection is optimized as a 384-well luciferase knockdown, the biologically relevant phenotypic analysis can proceed using the best siRNA transfection conditions. This approach provides a key technology for 384-well assay development when direct measurement of mRNA knockdown is not possible. It also allows for direct comparison of siRNA activity across cell lines from almost any mammalian species. Defining optimal conditions for siRNA delivery into mammalian cells will greatly increase the speed and quality of large-scale siRNA screening campaigns.     

Melanocortin 4 receptors interact with antimicrobial frog peptide analogues

We have developed fluorescence polarization (FP) assays of human melanocortin 4 receptor (MC4R) in 384-well microtiter plates using TAMRA-NDP-MSH as a tracer. The rank order of potency of agonists and antagonists agrees well relative to the published assays: SHU9119>MTII>NDP alphaMSH>alphaMSH. We have screened libraries of Korean plant extracts and frog peptide analogues in search of MC4R ligands using FP assays and cell-based CRE luciferase reporter assays. We report that FLGFLFKVASK, FLGWLFKVASK, FLGALFKWASK, and FLGWLFKWASK are the peptide analogues, which bind to human MC4R receptor with good affinity in vitro. FLGWLFKVASK and FLGWLFKWASK stimulated CRE-driven reporter gene via MC4R. In luciferase reporter assays, they possess the pharmacological and functional profiles of full agonists. We demonstrate the interaction of MC4R with 11-residue antimicrobial peptides derived from the Korean frog, Rana rugosa. The results suggest that MC4R interacts promiscuously with bioactive analogues of antimicrobial peptide, gaegurin-5.     

Use of a dual firefly and Renilla luciferase reporter gene assay to simultaneously determine drug selectivity at human corticotrophin releasing hormone 1 and 2 receptors

The aim of this study was to investigate and validate the use of a dual glow-signal luciferase reporter gene assay to simultaneously evaluate drug activity at two different seven-transmembrane receptor subtypes. Stable cell lines (CHO) transfected with either human corticotrophin releasing hormone 1 (hCRH1) receptors and a firefly luciferase reporter gene or hCRH2 and a Renilla luciferase reporter gene were created to provide different luciferase readouts for CRH1 and CRH2 receptors, respectively. Cells were combined for stimulation and measurement of luciferase luminescence in a 96-well plate format assay. The nonselective CRH agonists rat/human CRH and sauvagine caused concentration-dependent increases in luminescence via activation of CRH1 (firefly luciferase; pEC50 = 8.40 +/- 0.06 and 8.39 +/- 0.08, respectively, n = 8) and CRH2 (Renilla luciferase; pEC50 = 8.89 +/- 0.14 and 8.92 +/- 0.13, respectively, n = 8) receptors. The nonselective CRH antagonist astressin blocked these agonist-induced increases in luciferase at both CRH1 and CRH2 receptors. The selective CRH1 antagonist CP154,526 blocked r/hCRH- and sauvagine-induced increases in luciferase at CRH1 receptors only. These data report the expected pharmacology for CRH1 and CRH2 receptors. This assay enabled two receptor subtypes to be studied simultaneously in the same 96-well plate and generated robust data with low variability. It has the potential advantage of significant time and cost savings, with application to both basic research and compound screening.     

High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.