Abstract
Examining inflammasome-associated speck structures is one of the most preferred and easiest ways to evaluate inflammasome activation. Microscopy-based evaluation of specks is preferable, but this approach is time-consuming and limited to small sample sizes. Speck-containing cells can also be quantitated by a flow cytometric method, time of flight inflammasome evaluation (TOFIE). However, TOFIE cannot perform single-cell analysis such as simultaneously visualizing ASC specks and caspase-1 activity, their location, and physical characteristics. Here we describe the application of an imaging flow cytometry-based approach that overcomes these limitations. Inflammasome and Caspase-1 Activity Characterization and Evaluation (ICCE) is a high-throughput, single-cell, rapid image analysis utilizing the Amnis ImageStream X instrument with over 99.5% accuracy. ICCE quantitatively and qualitatively characterizes the frequency, area, and cellular distribution of ASC specks and caspase-1 activity in mouse and human cells.
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Acknowledgements
We thank Drs. Bibhuti Mishra and Kate Fitzgerald for their kind gift of immortalized BMDMs. We would also like to thank Richard De Marco for his training and instruction in using AMNIS IDEAS and assisting in the evaluation of our masking strategy. We would also like to thank the American Association of Immunologists (AAI) for the opportunity to present this method at IMMUNOLOGY 2017, Washington, D.C., May 12–16, 2017 [13]. Figure 1 was generated using Biorender (www.Biorender.com).
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Nagar, A., Harton, J.A. (2023). Flow Imaging of the Inflammasome: Evaluating ASC Speck Characteristics and Caspase-1 Activity. In: Barteneva, N.S., Vorobjev, I.A. (eds) Spectral and Imaging Cytometry. Methods in Molecular Biology, vol 2635. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3020-4_11
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DOI: https://doi.org/10.1007/978-1-0716-3020-4_11
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