Ovarian cancer is one of the most common gynecological cancers, which often leads to death due to late detection. Treatment is often unsuccessful in advanced stages due to resistance to platinum-based drugs (Pt-BM). A potential biomarker to predict Pt-BM resistance is ATP7A. Based on the determination of ATP7A, the treatment and survival of patients with ovarian cancer could be improved. However, the significance of determining ATP7A expression should be further evaluated. The aim of the thesis was to optimize the protocol for determining the expression of ATP7A by flow cytometry, which is a sensitive and powerful method. Until now, the expression of ATP7A in ovarian cancer cells has been determined by other methods, such as western blotting, qPCR and confocal microscopy. Since the transporter is mainly located inside the cell, for its detection it is necessary to carry out intracellular staining, which is time-consuming and technically demanding.
We were interested in whether there are differences in the level of ATP7A expression between cells that are first grown and stored frozen and then thawed before the experiment (hereafter "frozen cells") and cells that are first grown in cell culture and after peel them off, immediately use them for the experiment (hereinafter "growing cells"). The experiments were performed on the PEO1 cell line, which was isolated from a patient with advanced ovarian cancer. We performed two types of experiments, i.e. on frozen and growing cells. We found that prior freezing affects the permeabilization process in such a way that, during freezing, the membrane is most likely to be damaged and, therefore, perforation of the membrane occurs more quickly. Among the more important cell characteristics that affect the experiment is cell viability, which must exceed 90 %. An important factor influencing the results is also the experimenter. We also determined the optimal voltage on the cytometer, on which the signal strength depends. We found that the baseline voltage of the cytometer was optimal. Based on the average fluorescence intensity and the percentage of fluorescent events, we came to the conclusions that the results of the intracellular expression of ATP7A are not affected if the flow cytometry analysis is performed on frozen cells. The reproducibility of intracellular ATP7A expression results between and within experiments on growing and frozen cells separately was found to be acceptable. Based on the obtained results, we can conclude that in the future experiments could be performed on both frozen and growing cells. It would be optimal to make four parallel samples within the experiment and repeat the experiment four times.
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