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Portsmouth, United Kingdom

Glaysher S.,Translational Oncology Research Center
Methods in molecular biology (Clifton, N.J.) | Year: 2011

The ATP-based tumor chemosensitivity assay (ATP-TCA) is a standardised system which can be adapted to a variety of uses with both cell lines and primary cell cultures. It has a strong track record in drug development, mechanistic studies of chemoresistance and as an aid to clinical decision-making. The method starts with the extraction of cells in suspension from continuous cell culture (for cell lines), malignant effusions or biopsy material. Enzymatic digestion is used to obtain cells from tumor tissue. The assay uses a serum-free medium and polypropylene plates to prevent the growth of non-neoplastic cells over a 6-day incubation period followed by detergent-based extraction of cellular ATP for measurement by luciferin-luciferase assay in a luminometer. The assay results are usually shown as percentage inhibition at each concentration tested, and can be used with suitable software to examine synergy between different anticancer agents. Source


Glaysher S.,Translational Oncology Research Center
Methods in molecular biology (Clifton, N.J.) | Year: 2011

The preparation of cells from heavily contaminated tissue is challenging. It is usually best to avoid such specimens if possible, but for the study of colorectal and some other tumours, it is inevitable that this must be overcome. The best methods seem to use a combination of (1) debridement of necrotic or infected areas of the tumour, (2) enzymatic dissociation in the presence of antibiotics, (3) density centrifugation to remove debris, and (4) extensive washing of the tumour-derived cells prior to their use in cell culture methods. It is possible to obtain cells from up to 80-90% tumours with careful technique and cooperation from the clinical team. Source


Knight L.A.,Translational Oncology Research Center
Methods in molecular biology (Clifton, N.J.) | Year: 2011

Poor cell culture practice leads to poor science due largely to issues of cross-contamination between cell lines and of microbial contamination, but can be avoided by careful quality control and good laboratory practice. This chapter provides a brief and practical outline of the steps needed to mitigate the risks associated with poor cell culture practice. Good Laboratory Practice (GLP) is a set of principles that provides a framework within which laboratory studies are planned, performed, monitored, recorded, reported, and archived to ensure the reliability of data generated within a compliant laboratory. A key feature of this is the generation of quality-control methods and data management within the cell culture laboratory. Source


Cree I.A.,Translational Oncology Research Center
Methods in molecular biology (Clifton, N.J.) | Year: 2011

The basics of cell culture are now relatively common, though it was not always so. The pioneers of cell culture would envy our simple access to manufactured plastics, media and equipment for such studies. The prerequisites for cell culture are a well lit and suitably ventilated laboratory with a laminar flow hood (Class II), CO(2) incubator, benchtop centrifuge, microscope, plasticware (flasks and plates) and a supply of media with or without serum supplements. Not only can all of this be ordered easily over the internet, but large numbers of well-characterised cell lines are available from libraries maintained to a very high standard allowing the researcher to commence experiments rapidly and economically. Attention to safety and disposal is important, and maintenance of equipment remains essential. This chapter should enable researchers with little prior knowledge to set up a suitable laboratory to do basic cell culture, but there is still no substitute for experience within an existing well-run laboratory. Source


Harvey A.L.,University of Strathclyde | Cree I.A.,Translational Oncology Research Center
Planta Medica | Year: 2010

Natural products have been the biggest single source of anticancer drugs and there are continued efforts to explore the chemical diversity provided by nature in order to find new lead compounds. Bioassay test methods have developed into high throughput screening assays using both cell-based and molecular approaches. The various ways to detect effects on cell viability and cell proliferation are summarised and examples are given of developments using three-dimensional cultures and cancer stem cells. Cell-based reporter assays have also been created in order to look more directly for effects on specific physiological pathways. The molecular assays include those directed at microtubules and related proteins and at many different protein kinases. © Georg Thieme Verlag KG Stuttgart New York. Source

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