A fluorescence-based rapid screening assay for cytotoxic compounds

A simple fluorescence-based assay was developed for the rapid screening of potential cytotoxic compounds generated by combinatorial chemistry. The assay is based on detection of nuclear green fluorescent protein (GFP) staining of a human cervical cancer cell line (HeLa) carrying an integrated histone H2B-GFP fusion gene. Addition of a cytotoxic compound to the HeLa-GFP cells results in the eventual degradation of DNA and loss of the GFP nuclear fluorescence. Using this assay, we screened 11 distinct quinone derivatives and found that several of these compounds were cytotoxic. These compounds are structurally related to plumbagin an apoptosis-inducing naphthoquinone isolated from Black Walnut. In order to determine the mechanism by which cell death was induced, we performed additional experiments with the most cytotoxic quinones. These compounds were found to induce morphological changes (blebbing and nuclear condensation) consistent with induction of apoptosis. Additional tests revealed that the cytotoxic compounds induce both necrotic and apoptotic modes of death.     

A place for high-throughput electrophysiology in cardiac safety: screening hERG cell lines and novel compounds with the ion works HTTM system

Several commercially available pharmaceutical compounds have been shown to block the IKr current of the cardiac action potential. This effect can cause a prolongation of the electrocardiogram QT interval and a delay in ventricular repolarization. The Food and Drug Administration recommends that all new potential drug candidates be assessed for IKr block to avoid a potentially lethal cardiac arrhythmia known as torsades de pointes. Direct compound interaction with the human ether-a-go-go- related gene (hERG) product, a delayed rectifier potassium channel, has been identified as a molecular mechanism of IKr block. One strategy to identify compounds with hERG liability is to monitor hERG current inhibition using electrophysiology techniques. The authors describe the Ion Works HT instrument as a tool for screening cell lines expressing hERG channels. Based on current amplitude and stability criteria, a cell line was selected and used to perform a 300-compound screen. The screen was able to identify compounds with hERG activity within projects that spanned different therapeutic areas. The cell line selection and optimization, as well as the screening abilities of the Ion Works HT system, provide a powerful means of assessing hERGactive compounds early in the drug discovery pipeline.     

High-throughput RNAi screening in vitro: from cell lines to primary cells

Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale.     

In vitro cytotoxicity screening of water-dispersible metal oxide nanoparticles in human cell lines

In this study, we present in vitro cytotoxicity of iron oxide (Fe₃O₄) and manganese oxide (MnO) using live/dead cell assay, lactate dehydrogenase assay, and reactive oxygen species detection with variation of the concentration of nanoparticles (5–500 μg/ml), incubation time (18–96 h), and different human cell lines (lung adenocarcinoma, breast cancer cells, and glioblastoma cells). The surface of nanoparticles is modified with polyethyleneglycol-derivatized phospholipid to enhance the biocompatibility, water-solubility, and stability under an aqueous media. While the cytotoxic effect was negligible for 18 h incubation even at highest concentration of 500 μg/ml, MnO nanoparticle represented higher level of toxicity than those of Fe₃O₄ and the commercial medical contrast reagent, Feridex after 2 and 4 day incubation time. However, the cytotoxicity of Fe₃O₄ is equivalent or better than Feridex based on the live/dead cell viability assay. The engineered MnO and Fe₃O₄ exhibited excellent stability compared with Feridex for a prolonged incubation time.     

Method for screening ecdysone-inducible stable cell lines

Ecdysone-inducible systems are useful tools to study the function of various genes in different types of mammalian cells. However, it is technically difficult to establish stable cell lines. This is partly because the conditional expression system requires the expression of two or more components driven by different kinds of promoters. In this paper, we describe the use of a luciferase reporter gene system for rapid screening of cell lines that express the ecdysone and retinoid X receptors. Using this system, two human stable colon cancer cell lines, SW480/VgRXR and HCT116/VgRXR, were successfully generated. The expression of these receptors remained high after six months of continuous culturing. A tight regulation of gene induction in SW480/VgRXR was observed using 2 microM Ponasterone A. The gene induction was rapid and persistent. Our results demonstrated the advantage of establishing cell lines that continuously express high levels of ecdysone receptor proteins.     

A rapid and sensitive screening system for human type I collagen with the aim of discovering potent anti-aging or anti-fibrotic compounds

This study was undertaken with the aim of developing an easy and quick means of analyzing the effect of various compounds on the synthesis and secretion of human type I collagen at the protein level. A modification of the ELISA method was used on HFF-1 cells. For the proof of concept, we used thirteen compounds most of which are known to be antioxidants. Each compound was tested at concentrations of 0, 10 and 100 microM on HFF-1 cells for 24 h. Thirteen sets of experiments for each compound were performed in ANOVA with three replicates. Duncan multiple range test (DMRT) was used to compare the mean values obtained from the treatment groups. From the results it was concluded that Vitamin C, undecylenic acid, conjugated linoleic acid, glycolic acid, and citric acid at 100 microM concentration could be used for anti-wrinkling or protection from premature aging, which requires enhancement of collagen synthesis. Lactic acid, EGCG, resveratrol, and retinol that can inhibit collagen synthesis effectively in a dose-dependent manner may be used for anti-fibrosis treatment purposes.     

A multipumping flow system for in vitro screening of peroxynitrite scavengers

Peroxynitrite anion is a reactive nitrogen species formed in vivo by the rapid, controlled diffusion reaction between nitric oxide and superoxide radicals. By reacting with several biological molecules, peroxynitrite may cause important cellular and tissue deleterious effects, which have been associated with many diseases. In this work, an automated flow-based procedure for the in vitro generation of peroxynitrite and subsequent screening of the scavenging activity of selected compounds is developed. This procedure involves a multipumping flow system (MPFS) and exploits the ability of compounds such as lipoic acid, dihydrolipoic acid, cysteine, reduced glutathione, oxidized glutathione, sulindac, and sulindac sulfone to inhibit the chemiluminescent reaction of luminol with peroxynitrite under physiological simulated conditions. Peroxynitrite was generated in the MPFS by the online reaction of acidified hydrogen peroxide with nitrite, followed by a subsequent stabilization by merging with a sodium hydroxide solution to rapidly quench the developing reaction. The pulsed flow and the timed synchronized insertion of sample and reagent solutions provided by the MPFS ensure the establishment of the reaction zone only inside the flow cell, thus allowing maximum chemiluminescence emission detection. The results obtained for the assayed compounds show that, with the exception of oxidized glutathione, all are highly potent scavengers of peroxynitrite at the studied concentrations.     

A novel family of retroviral vectors for the rapid production of complex stable cell lines

The production of stable cell lines is an important technique in cell biology, and it is often the rate-limiting step in studies involving the characterization of the function of novel genes or gene mutations. To facilitate this process, a novel family of retroviral vectors, the pE vector family, has been generated. The retroviral sequences in the pE vectors have been taken from the Moloney murine leukemia virus (MMLV) vector pMFG, which has been shown to express cDNA inserts more consistently and at higher levels than earlier generations of MMLV vectors. These vectors contain four different internal ribosome entry site-selectable markers, allowing high-efficiency selection of transductants expressing the desired cDNA. The pE vectors have an episomal design to allow long-term production of high-titer virus without the need for subcloning the producer line. Using a strategy of combinatorial infection followed by combinatorial drug selection, we demonstrate that the pE vectors can be used to generate stable, polyclonal cell lines expressing at least three novel cDNAs in less than 2 weeks. The use of these vectors will thus dramatically accelerate the production of complex stable cell lines.     

A rapid screening method for detecting active compounds against erythromycin-resistant bacterial strains of Finnish origin

A rapid and simple microdilution technique on 96-well microplate based on turbidimetry was optimized and validated for screening of antimicrobial activity against erythromycin-resistant bacterial strains of Streptococcus pyogenes and Staphylococcus simulans isolated from Finnish patients. Using S. pyogenes ATCC 12351 as reference strain the developed method was evaluated by reproducibility measurements and using parameters typically employed for screening methods, i.e. signal-to-background, signal-to-noise and a screening-window coefficient, the Z' factor. The method was further used for screening a group of natural compounds and their synthetic derivatives against resistant bacterial strains. Of these, octyl and dodecyl gallates, and usnic and ursolic acids were the most active. The described method is a rapid, homogeneous, cost-effective and easy-to-perform system for screening of new potential antimicrobial agents in drug discovery.     

A microfluidic cell chip for virus isolation via rapid screening for permissive cells

Virus identification is a prerequisite not only for the early diagnosis of viral infectious diseases but also for the effective prevention of epidemics. Successful cultivation is the gold standard for identifying a virus, according to the Koch postulates. However, this requires screening for a permissive cell line, which is traditionally time-, reagent- and labor-intensive. Here, a simple and easy-to-operate microfluidic chip, formed by seeding a variety of cell lines and culturing them in parallel, is reported for use in virus cultivation and virus-permissive host-cell screening. The chip was tested by infection with two known viruses, enterovirus 71 (EV71) and influenza virus H1N1. Infection with EV71 and H1N1 caused significant cytopathic effects (CPE) in RD and MDCK cells, respectively, demonstrating that virus cultivation based on this microfluidic cell chip can be used as a substitute for the traditional plate-based culture method and reproduce the typical CPE caused by virus infection. Using this microfluidic cell chip method for virus cultivation could make it possible to identify an emerging virus in a high-throughput, automatic, and unprecedentedly fast way.     

The in vitro effect of DDT and related compounds on the succinoxidase system of rat heart

DDT and several of its related compounds were found to be inhibitory to rat heart succinoxidase at concentrations of from 10^?4 to 10^?5M, the degree of inhibition being about 70–90% at the higher concentrations. This inhibition could also be shown for cytochrome oxidase, but not for succinic dehydrogenase. The inhibition was demonstrable when the insecticide or its derivatives were added to the enzyme system as alcoholic solutions. Cholesterol at the same concentrations did not inhibit succinoxidase.When DDT was present in an oil emulsion, only a slight inhibition could be shown toward the succinoxidase system, even though the concentration in the emulsion was 10 times that of the highest level in alcohol.DDA, the acetic acid derivative of DDT, and a known metabolite thereof, was much less inhibitory toward the succinoxidase and cytochrome oxidase systems, even at 5 × 10^?4M.     

A human islet cell culture system for high-throughput screening

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds.     

A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro

Summary Many research efforts require the accurate determination of cell density in vitro. However, physical cell counting is inaccurate, time-intensive and requires removal of the cells from their growth environment, thereby introducing a host of potential artifacts. The current studies document a very simple method of determining cell density in microtiter wells via DNA-enhanced fluorescence. Fixed cells are stained with the A-T intercalating DNA stains DAPI or Hoechst 33342 and then fluorescence is quantified in a plate fluorometer. Fluorescence is shown to be linearly related to cell density as determined by two physical counting methods. The validity of the method is established in determining serum-stimulated growth of smooth muscle cells and in mitogen-induced growth of endothelial cells. The fixed cells can be stored for prolonged periods, thus allowing time-course proliferation assays without interassay variations. The fixed cells are also suitable for determinations of antigens of interest by ELISA. This method is potentially valuable in many in vitro systems where the quantification of cell density and proliferation is necessary. This work supported in part by NIH Cardiovascular Training Grant HL07423 and a grant from the American Federation for Aging Research to T. M. and HL35724 to B. W. EDITOR’S STATEMENT The technique described in this paper represents an approach to quantifying cell density in adherent monolayers of cultured cells in microtiter wells that is rapid and simple and does not require radioisotopes or removal of cells.     

Mammalian cell display for rapid screening scFv antibody therapy

Human antibodies are beginning to draw attention for use in immune gene therapy. The efficient generation of effective therapeutic monoclonal antibodies suitable for the treatment of cancers and infectious diseases would be enormously valuable. Antibody display methods are increasingly used to screen human monoclonal antibodies. Here we report the construction of a mammalian cell display method derived from a naive antibody repertoire, for which human single- chain variable fragments （scFv） have been transiently dis- played on 293T cell surfaces based on a pDisplay vector. The sizes of the current pDisplay-scFv antibody repertoires have been estimated to be 0.74 × 10^7. An immunoblot assay confirmed the expression of the scFv antibody library. The subceilular distribution of ErbB3-scFv expression plasmid facilitated the display of ErbB3 scFv on the cell membrane surface and the efficiency of the display was evaluated by fluorescence-activated cell sorting. This method of mamma- lian cell display was verified by successfully screening ErbB3 scFv candidates. A published scFv control was used to confirm the feasibility of the ErbB3 scFv screening process. Three ErbB3 scFv candidates were produced and they were found to have affinity similar to the published scFv candidate. Thus, the present screening system provided an optimal alternative for rapid acquisition of a novel candi- date scFv sequence to target genes with high affinity in vitro.