Abstract
The present protocol introduces a live-cell imaging of secretion activity (LCI-S) that is useful to visualize the real-time release of molecules from individual cells using an immunoassay coupled with total internal reflection fluorescence (FL) microscopy. This novel “live”-cell imaging technique has helped uncover the dynamics of regulated cell “death” by using this new approach. This protocol can observe the final stages of the regulated cell death process via single-cell imaging by targeting the extracellular release of damage-associated molecular patterns (DAMPs) from the cells expressing fluorescence resonance energy transfer (FRET) biosensors, such as a sensor for MLKL activation by RIPK3 based on FRET (SMART) and a sensor for caspase-1 activation based on FRET (SCAT1), which specifically identify the occurrence of regulated cell death processes.
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Acknowledgments
This work was supported in part by funding from JSPS KAKENHI (JP15H01366 and JP17H05496 to Y.S.), JST PRESTO (grant no. JP17940748 to Y.S.), and The Naito Foundation (to M.Y.). We would like to thank all the following people: Masayuki Miura of The University of Tokyo and Yoshifumi Yamaguchi of Hokkaido University for providing of SCAT1-expressed macrophages for IL-1β burst imaging , Hiroyasu Nakano and Shin Murai of Toho University School of Medicine for providing the SMART /HMGB1-mCherry-expressed L929 cells for HMGB1 burst imaging , Sotaro Uemura of The University of Tokyo and Kazuyo Moro of Osaka University School of Medicine for discussing LCI-S applications.
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Yamagishi, M., Shirasaki, Y. (2021). Live-Cell Imaging Technique to Visualize DAMPs Release During Regulated Cell Death. In: Kim, SB. (eds) Live Cell Imaging. Methods in Molecular Biology, vol 2274. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1258-3_28
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DOI: https://doi.org/10.1007/978-1-0716-1258-3_28
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Online ISBN: 978-1-0716-1258-3
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