Protocol for high-throughput identification of genes involved in pyroptotic cell death by live microscopy.

Intracellular lipopolysaccharide activates caspase-4/5 triggering pyroptotic cell death. We present a protocol for a high-throughput loss-of-function screening approach to identify interferon-gamma-stimulated genes regulating this pathway. We detail procedures for cell reverse-transfection with a custom-made small interfering RNA (siRNA) library in 384-well plates and priming with interferon-gamma. We then describe steps for transfection with lipopolysaccharide to induce pyroptosis and live-cell fluorescence microscopy using the IncuCyte S3 system to quantify cell death in real time without fixation. This approach enables reliable, high-throughput screening of cell death.