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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.

Author information

1
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA. Electronic address: martha.obrien@promega.com.
2
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA.
3
Dept. of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
4
Dept. of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.
5
Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA 93401, USA.

Abstract

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.

KEYWORDS:

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

PMID:
28268194
DOI:
10.1016/j.jim.2017.03.004
[Indexed for MEDLINE]
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