Abstract:

Objective: The aim of this study is to test the apoptotic response of the HT22 mouse hippocampal cell line, which is frequently used in neurobiology studies, to a standard differentiation medium at different times in order to understand its usability as a neuronal differentiation model. Methods: For seeding and culturing HT22 cells aside the differentiation, HG-DMEM and for differentiation B27+ supplemented NB+ medium were used respectively. A total of 9 different groups, including the control group, were cultured in standard cell culture environment. At the end of the experiment, the rates of early/late apoptosis and necrosis were determined with flow cytometric analysis of AnnexinV/PI labeled HT22 cells. After testing whether the raw data fit the normal distribution or not by employing Shapiro-Wilks test, further statistical analyzes were performed with the most appropriate test parametric or non-parametric tests. Results: Statistically significant differences were found in terms of early apoptosis (P<0,01) and late apoptosis (P<0,001) cell population rates, while there was no significant difference of necrosis (P=0,064). Significant (P<0,001) increase in early apoptosis was evident in the majority of groups cultured differentiation medium for 48 or 72 hours, compared to the cells cultured with only HG-DMEM or differentiation media for 24 hours. While a significant increase in late apoptosis was observed in the majority of the groups cultured with the differentiation medium compared to the control group (P<0.001), no significant difference was observed between the groups differentiated for different durations (P>0,05). Conclusion: HT22 hippocampal cell line, which is frequently used in in-vitro modeling of important pathobiological features of neurodegenerative diseases, but poorly understood for the suitability in neuronal differentiation, has a markedly increased response to a widely used differentiation medium in terms of early apoptosis (culturing duration dependent) and late apoptosis (regardless of duration). Our findings provide important clues at the level of apoptosis in terms of the usability of the HT22 cell line in differentiation studies.

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