Comparison of Methods in Studies of Cell Death Mechanisms

While studying the influence of ionizing radiation or certain chemical agents on cells, it is crucial to not only determine cytotoxicity, but also to follow cell death mechanisms. There are different methods to screen processes of cell death and still very important question remains unanswered about differences in results that could be caused by various experimental steps in procedures. Based on literature review two protocols of cell death determination were compared. First protocol regarded collecting cells floating in medium before trypsinization and following centrifugation of them. In the second protocol floating cells were discarded and attached ones were stained and fixed. In all experiments three different untreated cell lines (A172, DU145 as cancer cell lines and in comparison, fibroblasts (FB CCL 110), as a non- cancerous cell line) were used to test applied protocols. Cells were cultured and death processes were examined at different time points up to 120 h. Compared protocols showed statistically significant differences, especially in terms of necrosis, which was higher when included floating cells from culture medium and then centrifuging them. Therefore, presented results show importance of choosing a valid experimental procedure in case of evaluating cells viability and types of cell death pathways quantitatively.