Date of Award

1-2014

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

Elena Petroff

Committee Member

Vladislav Snitsarev

Committee Member

Judith Gorski

Subject(s)

Glioblastoma multiforme, Astrocytes, Apoptosis, Hydrogen-ion concentration

Abstract

Glioblastoma Multiforme (GBM) is the highest-grade glioma and the most malignant form of astrocytoma (Brat and Mapstone, 2003; Hjelmeland and others). Diagnosis of GBM carries a prognosis of less than 14 months after diagnosis, despite surgical intervention, radiation, and chemotherapy (Hjelmeland and others). Unfortunately, the prognosis of GBM has remained unchanged over the last thirty years, even with abundant resources devoted to research (Barres, 2008). GBM tumors have a low intracellular pH that results in increased levels of proliferation and decreased levels of apoptosis. As a result, astrocytes, the cell type responsible for GBM, have been cultured and evaluated to understand the behavior of GBM astrocytes.

Previous data shows that astrocytes cultured at pH 7.0 for several days will have higher cell numbers than those grown at the physiological pH 7.4 (Guercio, 2012). In these studies cell numbers were evaluated using manual counting to compare differences between culture conditions. A more automated approach that can evaluate multiple parameters on the same set of cells would allow for better understanding of the changes in proliferation and apoptosis in cultured cells. This would allow for more rapid turnover and consistent evaluation from culture to culture. Here we evaluated whether Promega’s ApoTox-Glo™ Triplex assay would produce reliable and repeatable results using cultured astrocytes. This assay evaluates levels of viability, cytotoxicity, and apoptosis in same set of cells.

Adult and Embryonic day 18 (El8) cells were harvested and cultured at either pH 7.0 or pH 7.4 for several days. Following the culture period, viability, cytotoxicity, and apoptosis levels were evaluated using the ApoTox-Glo™ Triplex Assay. Three different runs were completed and the results between runs varied, yet the results within runs were very consistent.

Viability or the number of live cells varied for each run of the assay with no consistent trends seen. Cytotoxicity or number of dead cells also varied between runs with El 8 cells, but adult cells showed a consistent trend of increased cytotoxicity at pH 7.4. Similar to viability, apoptosis levels showed varied results but with high variability from run to run and again, no consistent trends seen.

Although the results varied from run to run, results within each run were consistent. This suggests that this assay can be used with astrocytes and that further evaluation with different toxins that change viability, cytotoxicity, and apoptosis should be used to test the sensitivity of the kit. In addition to further work up with astrocytes, GBM cell lines should be evaluated to determine if this assay could be used to evaluate changes in viability, cytotoxicity, and apoptosis with novel treatments.

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