Hasc 71

RPMs were seeded at the desired concentration (0.4 × 10⁶ or 1.2 × 10⁶ cell/mL) onto coverslips with RPMI media with 5% FBS. After being activated with 5 μM nigericin, 3 mM ATP, cells were fixed in 2% paraformaldehyde. Cells were blocked with autologous serum and stained with primary anti-ASC (HASC-71, BioLegend) and secondary donkey anti-mouse AlexaFluor488 (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) and mounted on slides with DAPI-containing mounting medium (Prolong diamond antifade, Life Technologies). Images were acquired with an Eclipse Ti microscope (Nikon, Tokyo, Japan) equipped with a 10× (numerical aperture, 0.30) or 20× S Plan Fluor objective (numerical aperture, 0.45) and a digital Sight DS-QiMc camera (Nikon) and 387 nm/447 nm and 482 nm/536 nm filter sets (Semrock, Lake Forest, IL, USA).

To further confirm the ASC specks formation, we performed confocal microscopy as described previously (Micco et al, 2016 (link)). Briefly, PMA-primed WT and RNH1-KO THP1 cells were seeded on coverslips in 12-well plates before inflammasome activation. Next day, cells were stimulated for 1 h with Nigericin (5 μM) and fixed with 4% (vol/vol) PFA (Applichem) for 20 min at RT. After fixation, cells were washed three times with 1× PBS for 5 min and permeabilized with 0.1% Triton X-100 in 1× PBS for 15 min at RT. Cells were washed with 1× PBS and blocked with 4% (w/vol) BSA for 30 min at RT. After blocking, cells were incubated overnight at 4°C with the primary antibodies against ASC (HASC-71; BioLegend). Cells were washed three times with a washing buffer (1× PBS and 0.025% of Tween20) for 5 min and incubated for 1 h at RT with the Alexa Fluor secondary antibodies (Thermo Fisher Scientific). Cells were washed twice with a washing buffer for 5 min and incubated in with DAPI (1 μg/ml) for 1 min. Again, cells were washed, and coverslips were mounted on glass slides using mounting media (ProLong Gold Antifade). Images were captured with inverted confocal microscope LSM 710 (Zeiss) and analysed with ImageJ across at least 10 fields from three independent experiments.