Lipofectamine p300

To evaluate the presence of ASC oligomers, cells were transfected with the pLEX-MSC-ASC-GFP plasmid, a gift from Christian Stehlik (Addgene plasmid #73957; http://n2t.net/addgene:73957; RRID:Addgene_73957) [36 (link)]. Cells were seeded in 8-well LabTek removable chamber slides (Thermo Scientific, 177402) to 60%–70% of confluence. Transfections were conducted using Lipofectamine P300 (Invitrogen) in opti-MEM medium. Forty-eight hours after transfection, cells were exposed to the corresponding treatment. After treatment, cells were incubated with the CellMask™ Orange plasma membrane stain (1:10,000; Thermo Fisher Sci., C10045) and fixed with 4% PFA. The formation of ASC specks was assessed as the formation of reticulated structures, visualized with a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems, Spain) using a 63 × /1.32–0.60 oil PH3 CS objective and a confocal pinhole set at 1 Airy unit. From each well, three non-overlapping images from the top, middle and bottom areas were randomly taken. The number of speck-positive cells was determined by manual counting using ImageJ [34 (link)].

To evaluate the presence of ASC oligomers, cells were transfected with the pLEX-MSC-ASC-GFP plasmid, a gift from Christian Stehlik (Addgene plasmid #73957; http://n2t.net/addgene:73957; RRID:Addgene_73957) [36 (link)]. Cells were seeded in 8-well LabTek removable chamber slides (Thermo Scientific, 177402) to 60%–70% of confluence. Transfections were conducted using Lipofectamine P300 (Invitrogen) in opti-MEM medium. Forty-eight hours after transfection, cells were exposed to the corresponding treatment. After treatment, cells were incubated with the CellMask™ Orange plasma membrane stain (1:10,000; Thermo Fisher Sci., C10045) and fixed with 4% PFA. The formation of ASC specks was assessed as the formation of reticulated structures, visualized with a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems, Spain) using a 63 × /1.32–0.60 oil PH3 CS objective and a confocal pinhole set at 1 Airy unit. From each well, three non-overlapping images from the top, middle and bottom areas were randomly taken. The number of speck-positive cells was determined by manual counting using ImageJ [34 (link)].