Abstract
In myocardial infarction (MI), a number of cardiomyocytes undergo apoptosis. These apoptotic cardiomyocytes are promptly engulfed by phagocytes. If the dead cells are not engulfed, their noxious contents are released outside, resulting in induction of inflammation. Therefore, the removal of these dead cells is necessary. However, the contribution of each phagocyte type to the removal of apoptotic cells in infarcted hearts remains unresolved. Here, we describe an in vitro protocol for a phagocytosis assay to compare the engulfment ability of cardiac macrophages and cardiac myofibroblasts.
Keywords: Phagocytosis assay, Myofibroblast, Engulfment, Apoptosis, Myocardial infarction
Background
It has long been believed that the apoptotic cells generated in failed hearts are eliminated by cardiac macrophages. However, we found that cardiac myofibroblasts, which are responsible for tissue fibrosis, also have the ability to engulf apoptotic cells after MI (Nakaya et al., 2017). The discovery prompted us to compare the engulfment ability of cardiac macrophages and cardiac myofibroblasts. Herein, we provide a detailed protocol for an in vitro phagocytosis assay to evaluate the extent of phagocytic engulfment.
Materials and Reagents
Equipment
Software
Procedure
Procedures A and B are also applicable to phagocytosis assay of necroptotic cells by cardiac myofibroblasts (Horii et al., 2017).
Data analysis
Notes
In our opinion, this protocol is applicable for measuring phagocytosis by other cell types as well. In fact, we measured phagocytosis of apoptotic thymocytes by NIH3T3 cells using the same protocol (Nakaya et al., 2006). It should be noted that the cell sizes of phagocytes and apoptotic cells have to be considered in phagocytosis assay. The size of apoptotic cells should be smaller than that of phagocytes.
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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