Tumour chemo sensitivity assays: an investigation into the susceptibility of cells to chemotherapeutics

Doctoral thesis English OPEN
Gill, SC (2009)

To evaluate and identify new candidate cancer drug targets, there is an ongoing need for a reliable, sensitive and quantitative assay that enables the analysis of larger numbers of compounds in preclinical research. This thesis has developed, and optimized a sensitive enzyme-release assay for monitoring natural cytotoxicity. It measures the release of the intracellular enzyme adenylate kinase into the culture supernatant after membrane rupture and is evaluated as an indicator of cell death. This assay was proven to correlate and compete with currently used methodologies for the assessment of cytotoxicity and with its superior sensitivity; convenience and in expense, it should be applicable to the study of other cytotoxicity reactions. The resulting ToxiLight® kit is now being sold world-wide and rapidly became the top selling product for Lonza Bio Science with many references to its use in publications. It was proven from this investigation that to truly comprehend the effect a cytotoxic drug has on cells, two assays are required in combination; one to measure cytotoxicity and a second to measure viability. The two most sensitive kits tested in this study, the ViaLight® Plus assay and the newly designed ToxiLight® assay were used in combination to monitor the effect of commonly used cytotoxicity drugs on melanoma cells. It was hoped to find both a sensitive and resistant cell line for further analysis by MALDI-MS.
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    2.4.4 Preparation of Necrotic Models

    2.4.4.1 Freeze-Thawing of Cells 2.5 Cell Number Dilutions 2.6 Measuring ToxiLight with/without Cells 2.7 Preparation of Standards

    2.7.1 ATP Standards

    2.7.2 Myokinase Standards 2.8 Measurement of Cell Viability

    2.8.1 Measuring Cell Permeability with Propidium Iodide

    2.8.2 Uptake of Trypan Blue

    2.8.3 Assays Used to Measure Cytotoxicity

    2.8.3.1 ViaLight Plus Assay

    2.8.3.2 ToxiLight Assay

    2.8.3.3 Cyto-Tox-ONE Assay

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