Measuring the Apoptotic Response of the HT22 Mouse Hippocampal Cell Line to Neuronal Differentiation Medium

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.

Keywords

• HT22
• hippocampus
• neuronal differentiation
• apoptosis
• flow cytometry

HT22 Fare Hipokampal Hücre Hattının Nöronal Farklanma Besiyerine Verdiği Apoptotik Tepkinin Ölçülmesi

Abstract

Amaç: Bu çalışmanın amacı nörobiyoloji çalışmalarında sıkça kullanılmakta olan HT22 fare hipokampal hücre hattının nöronal farklılaşma modeli olarak kullanılabilirliğinin anlaşılması için standart bir farklandırma besiyerine farklı sürelerde verdiği apoptotik cevabın sınanmasıdır. Gereç ve Yöntemler: HT22 hücrelerinin ekilmesi ve farklandırma dışında kültüre edilmesi için HG-DMEM farklandırma için ise B27+ katkılı NB+ medyumu kullanılmıştır. Kontrol grubu da dâhil olmak üzere toplam 9 farklı grup standart olarak kültüre edilmiştir. Deney sonunda AnnexinV/PI işaretlemesiyle erken/geç apoptozis ve nekrozis oranları akım sitometrik olarak belirlenmiştir. Verilerin normal dağılıma uyup uymadığı Shapiro-Wilks testi ile sınandıktan sonra en uygun testle istatistiksel analiz gerçekleştirilmiştir. Bulgular: Gruplar arasındaki karşılaştırmada erken apoptozis (P<0,01) ve geç apoptozis (P<0,001) hücre popülasyon oranları açısından istatistiksel olarak anlamlı bir fark tespit edilirken nekrozis açısından anlamlı bir fark olmadığı gözlenmiştir (P=0,064). 48 ve 72 saat farklandırma besiyerinde kültüre edilen grupların büyük çoğunluğunda, sadece HG-DMEM ile veya 24 saat farklandırılan hücrelere kıyasla istatistiksel olarak anlamlı düzeyde (P<0,001) erken apoptozis artışı olduğu görülmüştür. Farklandırma besiyeri ile kültüre edilen grupların çoğunluğunda kontrol grubuna kıyasla anlamlı düzeyde geç apoptozis artışı gözlenirken (P<0,001), farklı süreler farklandırılan gruplar arasında anlamlı bir fark gözlenmemiştir (P>0,05). Sonuç: Nörodejeneratif hastalıklara ait önemli patobiyolojik özelliklerin in-vitro düzeyde modellenmesinde sıkça kullanılan, fakat nöronal farklılaşma açısından uygunluğu yeterince bilinmeyen HT22 hippokampal hücre hattının, yaygın olarak kullanılan bir farklandırma besiyerine erken apoptozis (kültürleme süresine bağlı) ve geç apoptozis (süreden bağımsız) artışı şeklinde anlamlı tepkiler oluşturduğu anlaşılmıştır. Bulgularımız HT22 hücre hattının farklılaşma çalışmalarında kullanılabilirliği açısından apoptozis düzeyinde önemli ipuçları sunmaktadır.

Keywords

• HT22
• hipokampus
• nöronal farklanma
• apoptozis
• akım sitometri

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