Use of Flow Cytometry for Detection of Apoptotic Cell Death in Th17 Cells

Proceedings

Nalbant, Ayten¹ 

[1] Molecular Immunology Laboratory, Department of Molecular Biology and Genetics, Izmir Institute of Technology, Ä°zmir 35430, Turkey

关键词: Th17 cells;    apoptosis;    flow cytometry;   

DOI  :  10.3390/proceedings2251560

学科分类：社会科学、人文和艺术（综合）

来源: mdpi

【 摘 要 】

Flow cytometry (FC) is a powerful and reliable system for cell death studies. It allows to study the molecular changes in both the surface and cytoplasmic part of the cells. Depending on the laser range of flow cytometry, it is possible to collect a large number of information about a single cell by combining multiple labeling strategies. With these assets, flow cytometry allows scientists to accelerate their research. Flow cytometry is also widely used in clinical studies. Therefore, we used this powerful tool to study human T helper 17 (Th17) cell apoptosis. Newly discovered Th17 cells are important players of immune response regulation. They are also involved in different types of pathologies including autoimmune diseases and cancer. There are intensive data in the literature about molecules that are involved in Th17 differentiation signaling networks, but the apoptotic and survival molecular mechanisms of this cells are not fully understood yet. Therefore, apoptosis of Th17 cells were measured by Flow cytometry. Healthy human subjects have been invited to study with the informed consent which is approved by the ethics committee. Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from peripheral blood of healthy volunteers. Phenotypically characterized and sorted naïve CD4+ T cells from PBMC were cultured under Th17 polarizing conditions. IL-17, IL-22 or CCR6 molecules were used to monitor Th17 cells. Apoptotic cell death of Th17 cells were measured by plasma membrane changes and DNA fragmentation. During differentiation stages, plasma membrane changes were monitored by Annexin V concomitantly with 7AAD. At day 7, there were Annexin V positive cells in Th17 cells. Apoptotic cell death occurs through sequential events including caspase activation and DNA fragmentation. DNA fragmentation data showed that Th17 cells were not apoptotic compared to negative control cultures. This finding suggested that survival molecules of Th17 cells interferes with this apoptotic process.

【 授权许可】

CC BY