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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Gill, SC (2009)
Languages: English
Types: Doctoral thesis
Subjects:
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.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • 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
    • 2.8.3.4 WST-1 Assay
    • 2.8.3.5 XTT Assay
    • 2.8.3.6 MTS Assay
    • 2.8.4 Inducing Necrotic Cell Death
    • 2.8.5 Fluorescent Microscopy
    • 2.8.5.1 Acridine Orange/Ethidium Bromide
    • 2.8.5.2 JC-1
    • 2.8.4 Preparation of Chromium release Cytotoxicity Assay
    • 2.8.4.1 Tumour Therapy Procedure
    • 2.8.4.2 Immunotherapy with DISC/mGM-CSF
    • 2.8.4.3 Chromium Release Cytotoxicity Assay
    • 2.9 Proteomic Samples
    • 2.9.1 Sample Preparation
    • 2.9.2 Protein Micro Assay
    • 2.9.3 Control Samples Used in MALDI analysis
    • 2.9.3.1 QC Samples
    • 2.9.3.2 BSA Samples
    • 2.9.3.3 Blanks
    • 2.9.4 Sample Processing on the Xcise Robotic System
    • 2.9.4.1 ZipTip Metholodology
    • 2.9.4.2 Preparation of Calibrants
    • 2.9.4.3 MALDI-MS Set-up and Analysis
    • 2.9.4.4 Identification Using MALDI MS-MS
    • 2.9.5 Bioinformatics Analysis
    • 2.9.5.1 ANN Analysis Chapter 3 A Novel Bioluminescent Assay for Necrotic Cell Death
    • 3.1 Introduction
    • 3.2 Results
    • 3.2.1 Formulation and Measurement of AK
    • 3.2.2 Measuring AK within Cells
    • 3.2.3 ToxiLight With/Without Cells
    • 3.2.4 Suitability of the ToxiLight Assay Compared To
    • 3.2.4.1 Comparison with LDH Release
    • 3.2.4.2 Enzyme Stability 115
    • 3.2.4.3 Comparison with Propidium Iodide Uptake 117
    • 3.2.4.4 Comparison with Chromium51 Release 119
    • 3.2.4.5 Measuring Cell Viability 121
    • 3.2.4.6 Measuring ATP as a Cell Marker 125
    • 3.2.5 Optimisation of ToxiLight 127
    • 3.2.6 Combination of ToxiLight and ViaLight Plus Assays 131
    • 3.3 Discussion 133
    • 3.3.1 Optimisation of ToxiLight Assay 133
    • 3.3.2 ToxiLight Compared to Traditional Assays 133 Chapter 4 Utilising Bioluminescent Assays to Assess Cell Death in Melanoma Cells
    • 4.1 Introduction 138
    • 4.2 Results 144
    • 4.2.1 Measuring ATP and AK as a Cell Marker in Melanoma 146
    • 4.2.1.1 Effect of Chemo Toxic Agents on Assays 146
    • 4.2.1.2 Effect of Chemo Toxic Agents on Melanoma 148
    • 4.2.2 Effect of Trichostatin A on Melanoma Cells 162
    • 4.2.2.1 Effect of Trichostatin A on MEWO cells 163
    • 4.2.2.2 Effect of Trichostatin A on Ma Mel 28 cells 168
    • 4.2.2.3 Effect of Trichostatin A on Ma Mel 26a cells 173
    • 4.3 Discussion 178
    • 4.3.1 Effect of Doxorubicin of Melanoma cells 179
    • 5.3.2 Importance of QC in MALDI-MS analysis
    • 5.2.3 MALDI-MS analysis
    • 5.3.4 Analysis by ANNs
    • 5.3.5 Clinical relevance of identified ions
    • 5.3.6 Conclusion Chapter 6 Conclusions and Further Study
    • 6.1 ToxiLight and its Use as a Cytotoxicity Assay
    • 6.2 Cytotoxicity Assays and Melanoma Study
    • 6.3 The Use of Trichostatin A in the Treatment of Melanoma
    • 6.4 Use of Cancer Cell Lines in Proteomics
    • 6.5 Use of Bioinformatics as a Tool in Biomarker Identification
    • 6.6 Personalised Medicine References
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