title: A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.
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.
authors.affiliation: Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA. Electronic address: martha.obrien@promega.com.
authors.forename: Martha
authors.initials: M
authors.lastname: O'Brien
authors.affiliation: Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA.
authors.forename: Danielle
authors.initials: D
authors.lastname: Moehring
authors.affiliation: Dept. of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Raúl
authors.initials: R
authors.lastname: Muñoz-Planillo
authors.affiliation: Dept. of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Núñez
authors.affiliation: Dept. of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Callaway
authors.affiliation: Dept. of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Jenny
authors.initials: J
authors.lastname: Ting
authors.affiliation: Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA 93401, USA.
authors.forename: Mike
authors.initials: M
authors.lastname: Scurria
authors.affiliation: Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA 93401, USA.
authors.forename: Tim
authors.initials: T
authors.lastname: Ugo
authors.affiliation: Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA 93401, USA.
authors.forename: Laurent
authors.initials: L
authors.lastname: Bernad
authors.affiliation: Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA.
authors.forename: James
authors.initials: J
authors.lastname: Cali
authors.affiliation: Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Lazar
mesh_terms: D000818:Animals; D020170:Caspase 1 / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009000:Monocytes / Q000201:enzymology / Q000378:metabolism*; D000069292:Pyroptosis; D012680:Sensitivity and Specificity
chemical_list: D058847:Inflammasomes; D008156:Luciferases; D020170:Caspase 1
keywords: Bioluminescent; Caspase-1 assay; Inflammasome; Macrophages; Pyroptosis; THP-1 monocytes