Nucleopore polycarbonate

Unilamellar liposomes were prepared at room temperature (≈23°C) by hydration of lipid films and extrusion through polycarbonate filters with 0.1‐μm pore diameter (Nucleopore Polycarbonate, Whatman) according to the method described by Avanti Polar Lipids (www.avantilipids.com). The E. coli polar lipids and the extruding equipment used for liposome preparation were purchased from Avanti Polar Lipids. The liposomes were stored at −80°C in buffer solution (150 mM NaCl, 20 mM Hepes, pH 7.25). After incubation with 1 μM mM GSDMD and 0.2 μM caspase‐1 for 90 min at 37°C in buffer solution (50 mM NaCl, 100 mM Hepes, 5 mM TCEP, pH 7.4), the liposomes were adsorbed onto freshly cleaved mica in buffer solution (50 mM NaCl, 20 mM Hepes, pH 7.4) (Muller et al, 1997). After an adsorption time of 30 min, the sample was washed several times with the AFM imaging buffer (150 mM NaCl, 20 mM Hepes, pH 7.8) to remove weakly adsorbed protein. Buffer solutions were freshly made using nanopure water (18.2 MΩ cm⁻¹) and pro‐analysis (> 98.5%) purity grade reagents from Sigma‐Aldrich and Merck. Each experimental condition characterized by AFM was reproduced at least three times. Liposomes made from E. coli polar lipids incubated in buffer solution but in the absence of GSDMD showed no arc‐, slit‐ or ring‐like structures when imaged by AFM (Mulvihill et al, 2015).