RESUMO
We have developed a novel fluorescent Oxygen BioSensor technology platform adaptable to many applications in the area of drug discovery and development, particularly cell-based assays. This biosensor technology requires no additional reagents or incubations, and affords continuous real-time readout of dissolved oxygen concentrations. Since the level of oxygen dissolved in an assay's medium correlates to the number and viability of the cells in the medium, this technology is ideally suited for monitoring cell viability, proliferation, or death. The technology is particularly well suited to investigating cells' kinetic responses to proliferative or toxic stimuli, such as drugs. When incorporated into a 96- or 384-well microplate format, it is compatible with standard laboratory automation systems. Here we present data illustrating the application of the Oxygen BioSensor technology for rapid, homogeneous detection and evaluation of metabolic activity of a variety of eukaryotic and prokaryotic cells, including mammalian cells, insect cells, yeast, and bacteria. In the absence of toxic substances, we find a good correlation between cell number and signal over a wide range of cell concentrations and growth times. To evaluate the usefulness of the Oxygen BioSensor for cytotoxicity assays, we have performed a series of experiments using a range of toxic agents and cell types, including both bacteria and mammalian cell lines. In a side-by-side comparison to standard MTT assays using HL60 cells, comparable IC(50) values were found with the Oxygen BioSensor for five different toxins or drugs. This assay method does not have the need for additional reagents, handling steps, or incubation periods required by the MTT assays.
Assuntos
Técnicas Biossensoriais , Espectrometria de Fluorescência/métodos , Animais , Antibacterianos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Corantes Fluorescentes , Humanos , Testes de Sensibilidade Microbiana , OxigênioAssuntos
Anticorpos Antivirais , Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Transformação Celular Viral , Vírus 40 dos Símios/imunologia , Animais , Complexo Antígeno-Anticorpo , Antígenos Virais de Tumores , Mapeamento Cromossômico , Humanos , Células Híbridas/imunologia , Camundongos , Peso MolecularAssuntos
Antígenos de Neoplasias/análise , Antígenos Virais/análise , Transformação Celular Viral , Vírus 40 dos Símios/imunologia , Proteínas Virais/imunologia , Animais , Linhagem Celular , Precipitação Química , Camundongos , Peso Molecular , Mutação , Peptídeos/imunologia , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/crescimento & desenvolvimentoRESUMO
Administration of estradiol-17beta to male Xenopus laevis induces synthesis and secretion by the liver of the egg yolk precursor protein vitellogenin. RNA extracted from livers of estradiol-17beta-treated Xenopus laevis directs the synthesis of the entire 200,000-dalton vitellogenin monomer in a cell-free protein synthesizing system derived from rabbit reticulocytes. Vitellogenin synthesized in vitro was isolated and quantitated by indirect immunoprecipitation and identified by comparison to authentic [14C]vitellogenin. The in vitro product and [14C]vitellogenin co-migrate on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and they exhibit identical immunoprecipitation curves. Xenopus laevis vitellogenin messenger RNA has a sedimentation coefficient of approximately 30 S in sucrose density gradients. It can be purified approximately 60-fold from cell RNA by poly(U)-Sepharose chromatography and therefore appears to contain a polyadenylate sequence. Vitellogenin mRNA and vitellogenin synthesis in vivo could not be detected in unstimulated male Xenopus laevis. The relative rate of vitellogenin synthesis and the level of vitellogenin mRNA were determined at various times following the administration of estradiol-17beta. Vitellogenin synthesis is maximal 12 days after estradiol-17beta administration when it comprises approximately 70% of cell protein synthesis. The level of vitellogenin mRNA and the intracellular rate of vitellogenin synthesis exhibit a close correspondence from 4 to 16 days after administration of estradiol-17beta.
Assuntos
Estradiol/farmacologia , Lipoproteínas/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Vitelogeninas/biossíntese , Animais , Especificidade de Anticorpos , Técnicas In Vitro , Fígado/metabolismo , Masculino , Peso Molecular , Testes de Precipitina , Biossíntese de Proteínas/efeitos dos fármacos , RNA/metabolismo , RNA Mensageiro/isolamento & purificação , Coelhos , Reticulócitos/metabolismo , Vitelogeninas/imunologia , XenopusAssuntos
Formação de Anticorpos , Herpesviridae/imunologia , Mononucleose Infecciosa/imunologia , Ativação Linfocitária , Anticorpos/isolamento & purificação , Antígenos Virais , Linhagem Celular , Imunofluorescência , Herpesvirus Humano 4/imunologia , Humanos , Masculino , Métodos , Testes de Neutralização , Fatores de TempoRESUMO
Blood leukocytes of two species of new world primates, other than human, transform following exposure to Epstein-Barr virus. The transformed simian cells produce Epstein-Barr virus antigens and infectious (transforming) virus. The simian lymphoblastoid cells form multinucleate giant cells that appear to be selective sites for the production of Epstein-Barr virus. Multinucleate cells reveal intranuclear inclusions; in both species, a large proportion of giant cells contain Epstein-Barr virus antigen detectable by immunofluorescence.
Assuntos
Fibroblastos/metabolismo , Haplorrinos/imunologia , Herpesviridae/patogenicidade , Leucócitos/metabolismo , Animais , Reações Antígeno-Anticorpo , Antígenos Virais/biossíntese , Bovinos , Linhagem Celular , Transformação Celular Neoplásica , Células Cultivadas , Cromossomos/patologia , Testes de Fixação de Complemento , Efeito Citopatogênico Viral , Feto , Imunofluorescência , Herpesvirus Humano 4/patogenicidade , Humanos , Soros Imunes , Rim , Leucócitos/imunologia , Patologia , PlacentaAssuntos
Transformação Celular Neoplásica , Técnicas de Cultura , Herpesviridae/imunologia , Leucócitos , Anticorpos , Formação de Anticorpos , Antígenos Virais , Linhagem Celular/microbiologia , Linhagem Celular/efeitos da radiação , Células Cultivadas , Feminino , Imunofluorescência , Herpesvirus Humano 4/imunologia , Humanos , Soros Imunes , Cariotipagem , Leucócitos/imunologia , Leucócitos/microbiologia , Leucócitos/efeitos da radiação , Masculino , Métodos , Mycoplasma , Testes de Neutralização , Efeitos da Radiação , Fatores de TempoRESUMO
A method was devised for obtaining single cell clones of human leukocytic cell lines in the presence of a human placental cell feeder layer. Clones of two lines, LS-B and EB(3), which contain Epstein-Barr viral (EBV) antigen in approximately 1% of cells were tested for EBV antigen. Since all EB(3) clones and LS-B clones contained EBV antigen, it is concluded that in vitro EBV genome is associated with all of the cells.
