Hcx pl fluotar objective

Cells were seeded on coverslips (150 000 cells per coverslip) and treated as described above with the addition of 25 μM Z-VAD-fmk (Bachem) to prevent detachment of pyroptotic cells and therefore loss of cells with ASC specks. Coverslips were fixed with 4% paraformaldehyde (PFA) (15 min, 37 °C, Alfa Aesar) and washed with PBS. Nuclei were stained with Hoechst 33342 (Life Technologies) for 10 min and the slides mounted using Vectashield (Vector Laboratories). For quantifications of ASC aggregates (specks or filaments), ten random regions of interest were imaged at × 20 magnification (Leica DMI3000B inverted fluorescence microscope, HCX PL FLUOTAR objective, Leica DFC3000G camera and LAS AF Version 3 software) and the number of ASC aggregates and cells were counted. For representative images, the slides were imaged at × 63 magnification (Leica point scanning confocal ‘SP8', HC PL APO CS2 × 63 objective, Leica AF software version 3). For measurements of speck sizes, random regions of interest were images at × 63 magnification (Leica point scanning confocal ‘SP8') and the largest diameters of individual specks were measured using Fiji63 (link).