Promega GmbH

Zellbasierte Assays von A (Apoptose),  bis Z (Zytotoxizität)

Abstracts zu Promega-Seminaren

Oktober 2008

Caspase-Glo 3/7 Assay: Simple, sensitive and fast assay for detection apoptosis in cell cultures.
Mydlíková Zuzana
Cancer research Institute, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38 Bratislava, Slovakia

Apoptosis is a form of programmed cell death in mammalian cells. It involves a series of biochemical events leading to a characteristic cell morphology and death. Caspases plays essential roles of this biochemical events of apoptosis. Caspase 3 and caspase 7 are effector caspases with location at the end of the caspase cascade. Their function is in the activation of nucleases, which leads to DNA fragmentation. Caspase-3 and caspase-7 share common substrate specificity and structure. Both have substrate selectivity for the amino acid sequence Asp-Glu-Val-Asp (DEVD). The Caspase-Glo 3/7 assay provides a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity, luciferase activity and cell lysis. Adding a single Caspase-Glo 3/7 Reagent in an add-mix-measure format results in cell lysis, followed by caspase cleavage of the substrate and generation of a glow-type luminescent signal, produced by luciferase. Luminescence is proportional to the amount of caspase activity present. We used this very simple, sensitive and fast assay for detection of apoptosis in cell cultures of hamster ovary XPB deficient cells. Using this method we have succeded to identify the activation time of Caspase3 and 7 and based on the development of apoptosis after UV irradiation separate particular deficient cell lines.

Detection of apoptosis in leukemic cells L1210 using TUNEL assay
Kopásková Marcela¹, Hudecová Alexandra¹, Rauko Peter²
1 Department of Genetics, Faculty of Natural Sciences, Comenius University, Mlynská dolina B1, 842 15 Bratislava, Slovakia, kopaskova@fns.uniba.sk
2 Cancer research Institute, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38 Bratislava, Slovakia

Apoptosis or Programmed cell death exhibit several morphological changes, which are characterized by cell shrinkage, nuclear condensation, membrane blebbing, fragmentation into membrane bound apoptotic bodies. Cells undergoing apoptosis eventually cleave their DNA with nucleases in a characteristic pattern that leaves free 3'OH ends. This permits the identification of cells undergoing apoptosis by transfer of fluorescence-labeled dUTP to the free 3'OH ends using the enzyme terminal deoxynucleotidyl transferase (TdT) in a process termed terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL). The TUNEL assay is a sensitive method which is used for analyzing DNA fragmentation by labeling the 3´OH ends of DNA strand break. This method is based on the ability of terminal deoxynucleotidyl transfedase (TdT) to attach a fluorescein - conjugated deoxy-uracil to the 3´-OH end of cut DNA. The label incorporated at the damaged sites of the DNA is visualized by fluorescence microscopy. The advantages of the method include detection of very early stages of apoptosis at single cell level, requirement small numbers of cell per sample (<10 000), sensitivity for detecting DNA damage. Phytotherapy is one of the modern methods, which can induces injury the tumor cells, which subsequently die by a number of processes. One of those processes is apoptosis, and its measurement can be a useful tool for understanding the mechanisms of action of phytochemicals. The aimed of our research is to explore the effects of extract from Lilium candidum L. on the proliferation and apoptosis in leukemic cells L1210.

Mittwoch, 24. Mai 2006
Im Hörsaal des MPI für Experimentelle Medizin, Hermann-Rein-Straße 3, 37075 Göttingen

Dr. Gunnar Dietz, Universität Göttingen, Neurologische Universitätsklinik, Abt. Prof. Bähr: „Inhibition of neuronal cell death in vitro using cell-permeable fusion proteins“

When cerebellar granule neurons are deprived of serum and depolarizing potassium concentrations, they quickly undergo apoptosis. Moreover, they are killed by excessive glutamate treatment. The sensitivity of these neurons against potassium removal and glutamate has been used to elucidate the mechanisms of cell death in primary neurons in development and disease. Usually, neuronal survival is assessed by recording images of cultured cells after staining them with a fluorescent dye. Although subsequent cell counting is facilitated by image analysis software, it is time consuming and prone to errors in the detection of cerebellar granule neurons. We have recently been using the CellTiter-Blue™ Cell Viability Assay to measure cellular survival in cerebellar granule neurons and to examine the neuroprotective potential of various compounds, including proteins coupled to a domain that enhances their cellular uptake.

Herr Dr. Dudas, Universität Göttingen, Abteilung Gastroenterologie: „DeadEnd™ Colorimetric TUNEL System - eine gut kombinierbare und universal verwendbare Apoptose-Nachweis-Methode in kultivierten Zellen und im Gewebe“

Apoptose (programmierter Zelltod) ist ein genetisch regulierter aktiver biochemischer Prozess, der für Wachstum, Differenzierung und Aufrechterhaltung der Homöostase von Geweben und Organismen von zentraler Bedeutung ist. Apoptose unterscheidet sich von der Nekrose durch morphologische und biochemische Kriterien. Ein wichtiges biochemisches Merkmal der Apoptose ist die Aktivierung intrazellulärer Ca²⁺ - und Mg²⁺- abhängiger Endonukleasen (DNAsen), die zu einer Fragmentierung der DNA führt. Die DNAsen degradieren DNA in kleine 180-200 bp Fragmente. Neben morphologischen Methoden, ist der Nachweiß von DNA Fragmentierung eine der wichtigsten Methoden, um Apoptose in Geweben nachzuweisen. TUNEL - Test („terminal deoxy-Uridine-triphosphate nick end labeling“): Während der Apoptose wird die DNA durch DNAsen fragmentiert. Daraus resultiert eine Zunahme von freien 3’ - OH Enden. Man macht sich dieses zu Nutze, indem man durch eine Transferase an die jetzt vielfach vorhandenen freien 3’ - Enden ein mit Biotin - markiertes deoxi-Nukleotid-Diphosphat substituiert. Mit Peroxidase (POD) verknüpftes Streptavidin bindet an das Biotin. Nach POD-Substratzugabe kommt es zur selektiven Anfärbung in der Umgebung der fragmentierten DNA. Das Ergebnis der Methode ist eine Braunfärbung von apoptotischen Zellkernen im Gewebe. Der “Dead-End Colorimetric TUNEL System” zeichnet sich durch mehrere Vorteile aus:
1. Kolorimetrische Methode, keine Störung durch Autofluoreszenz
2. Das Avindin-Biotin System wird in der Methode verwendet und erlaubt eine spezifische Verstärkung der detektierten Signale
3. Die Proteinase K Permeabilisierung im Gewebe verbessert die Sensitivität
4. Das Biotin-Avidin System erlaubt eine Kombination mit anderen Methoden (z. B. Immunohistochemie)
5. Das Detektionssystem ermöglicht den Apoptosenachweis in kultivierten Zellen, in Kryostatschnitten und in Paraffin eingebetteten Geweben

 

Cell Titer-Glo Cell Viability Assay application for determination of cell proliferation and drug cytotoxicity

Jozefa Gadek-Wesierski, Institute of Cancer Research, University of Vienna, Vienna, AUSTRIA

Determination of the cytotoxic action of a variety of anti-cancer agents on distinct normal and malignant cells is an important challenge. However, to exactly characterize the mechanism of action, it is necessary to discriminate between direct and indirect cytotoxic effects. Therefore, methods allowing reproducible and reliable determination of the increased direct cytotoxicity as well as of the inhibition of cell proliferation are required. Moreover, a standardization of the method is necessary. We tested the employment of the CellTiter-Glo Assay for examination of the direct and indirect cytotoxicity after treatment of human cells with agents inducing cell cycle arrest, apoptosis or necrosis. We analysed the viability of adherent cells as well as detached cells by Trypan Blue staining and the CellTiter-Glo Assay and compared the results. Our data show that by using the CellTiter-Glo Assay viable cells can be clearly discriminated from dead cells. 

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