Abstract
In myocardial infarction (MI), a plenty of cardiomyocytes undergo necrosis and necroptosis due to the lack of oxygen and nutrients. The dead cardiomyocytes are promptly engulfed by phagocytes. When the dead cells are not engulfed, the noxious contents of the cells are released outside, and thus, induce inflammation, and obstruct the function of organs. Therefore, phagocytosis is crucial for maintaining homeostasis of organs. Herein, we describe a protocol of an in vitro phagocytosis assay of necroptotic cells.
Keywords: Phagocytosis assay, Myofibroblast, Engulfment, Necrosis, Myocardial infarction, Isolation
Background
Previously, necrotic and necroptotic cells were believed to be eliminated only by cardiac macrophages in failed hearts. However, we found that cardiac myofibroblasts, which are responsible for tissue fibrosis, engulf dead cells after an MI (Nakaya et al., 2017). Herein, we provide a detailed protocol for an in vitro phagocytosis assay of necroptotic cells, employing L929 cells that undergo necroptosis by TNF-α stimulation in a caspase-3 inhibitor, Z-VAD-FMK.
Materials and Reagents
Equipment
Software
Procedure
Procedures A and B are also applicable to the assay to determine phagocytosis of apoptotic cells by cardiac macrophages and Cardiac Myofibroblasts (Horii et al., 2017).
Data analysis
Recipes
Acknowledgments
When using this protocol, please refer to M Nakaya et al. (2017) J Clin Invest. Funding was provided by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) [to M.N (17H03984)]; from Grant-in-Aid for Scientific Research on Innovative Areas (Homeostatic regulation by various types of cell death) from MEXT [to M.N (17H05510)]. All animal experiments were performed using approved protocols and in accordance with the guidelines of Kyushu University.
References
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