Work

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

Year: 2026

Type: article

Abstract: 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.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2026.104884

Authors: Aleksandra Bajger, Lukasz S. Borowski, Roman J. Szczesny, Michal P. Wandel

Institutions: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Polish Academy of Sciences, University of Warsaw

Language: English

Topic: Inflammasome and immune disorders

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,550