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J Immunol Methods. 2017 Aug;447:1-13. doi: 10.1016/j.jim.2017.03.004. Epub 2017 Mar 6.

A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.

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Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA. Electronic address:
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA.
Dept. of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Dept. of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.
Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA 93401, USA.


Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1-/- mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators.


Bioluminescent; Caspase-1 assay; Inflammasome; Macrophages; Pyroptosis; THP-1 monocytes

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