Cells were seeded at a concentration of 2 × 105 cells/well onto round coverslips in 24-well plates. After treatment, cells were fixed in 4% paraformaldehyde for 30 min and blocked and permeabilized in 500 μl blocking buffer (PBS, 10% FBS, 0.5% Triton X-100) for 1 h at room temperature, before incubation with the primary antibody in blocking buffer overnight at 4 °C. After washing with PBS three times, cells were treated with a dilution of 1:200 of the Alexa-488 or Alexa-594 conjugated secondary antibody for 1 h. Nuclei were co-stained with 4’,6-diamidino-2-phenylindole (DAPI, from Cell Signalling Technology). The images were captured with a Zeiss LSM710 confocal fluorescence microscope with objective Plan-Apochromat 63×/1.40 oil DIC M27 objective. Z-stack digital images were acquired with ZEN imaging software (Carl Zeiss, GmbH). ASC specks were counted in five random areas of each image in triplicate experiments, ensuring a minimum of 100 cells from each treatment condition.
