Optimized assay for the measurement of caspase‐1 expression, release, and activity in murine macrophages

Abstract

Caspase‐1 is a central component of the cellular inflammatory response, in particular with respect to its key role in the activation of the NLRP3 inflammasome pathway. Activation of caspase‐1 ensures the cleavage and release of the pro‐inflammatory cytokines interleukin (IL)‐1β and IL‐18, as well as the initiation of pyroptosis, that is a distinct form of programmed cell death. Hence, a wide‐ranging analysis of caspase‐1 in cellular systems is of special interest. To meet this requirement, we improved a commercial luminescence‐based caspase‐1 activity assay and combined it with the determination of the expression and release of caspase‐1 protein, using murine J774A.1 macrophages as a model system for in vitro studies. The presented assay procedure offers additional options over commercially available caspase‐1 activity assays as it allows for: (i) The simultaneous analysis of caspase‐1 activity and protein expression (both intracellular as well as secreted protein in supernatant) out of the same cell sample. (ii) A more economical use of valuable compounds and materials and improves the informative value of each measurement, since all results are generated from the same cell sample. Our optimized assay is therefore suitable for an efficient and reliable screening of modulatory effects of compounds of interest at various regulatory stages of the caspase‐1 system.