Pyroptosis Detection via Multiplexing Cell Death and Bioluminescent Caspase-1 Activity Assessments
Poster Jul 14, 2017
Martha O’Brien1, Mike Scurria2, Tim Ugo2, Laurent Bernad2, James Cali1, and Dan Lazar1
Pyroptosis is a unique immunogenic form of cell death dependent on inflammasome activation, provoked when pattern recognition receptors (PRRs) sense microbial infection or cellular stress. Caspase-1, an essential component of inflammasomes, mediates this innate immune response by processing cytokines and eliciting cell death. In addition to caspase-1, other inflammatory caspases (caspase-11 in mice and caspases-4, 5 in humans) can cleave gasdermin D and elicit pyroptosis in response to some intracellular microbial infections. However, caspase-1 is also activated under these circumstances, therefore caspase-1 activation coincident with cell death is a reliable indicator of pyroptosis. Understanding when PRR sensing leads to cell death and when it does not is essential to understanding the complexities of the innate immune response. We have developed a convenient method to monitor pyroptosis by multiplexing in the same assay well our homogeneous, bioluminescent caspase-1 activity assay with a membrane-impermeant, stable DNA dye for the detection of cell death. In contrast to endpoint assays for cell death such as lactate dehydrogenase (LDH) assays, the DNA dye can be used in real-time. Cell death can be monitored over time and when first detected, the caspase-1 assay can be performed confirming pyroptosis. Alternatively, culture medium can be removed for testing for caspase-1 release, leaving the cells available for measuring cell death by a variety of means. We show examples of pyroptosis in human THP-1 monocytes and mouse J774A.1 macrophages.
Multiplexing cell-based assays is possible using 3D culture models that are larger and more complex than monolayers
Real-time detection methods to measure live or dead cells provide much flexibility for multiplexing
All multiplexed assay combinations should be verified using appropriate controls for each 3D cell culture model.
Basic fibroblast growth factor (bFGF) is widely used in vitro for the maintenance and stimulation of a variety of cells. However, use of native bFGF in cell biology is limited by the fact that bFGF rapidly degrades at physiological temperatures. We have addressed this problem with an engineered form of bFGF, named Heat Stable bFGF (HS bFGF), which is stable at 37 degrees Celsius.READ MORE