Ultrapure endotoxin free 0.1 m pbs

EVs from Gsdmd^I105N/I105N, Gsdmd^−/−, WT BMDM and iBMDM cell culture supernatants were isolated using differential ultracentrifugation. Cell culture supernatant were spun at 3200×g for 10 min at 4 °C to remove cells and cell debris. The supernatant was transferred to 14 × 89 mm Beckman Ultra-Clear ultracentrifuge tubes and centrifuged at 10,000×g for 30 min (Beckman Coulter, CA USA) using a Beckman Coulter Optima XE-100 ultracentrifuge fitted with a SW41Ti Rotor (Beckman Coulter, CA USA), to collect large EV. Large EV pellets were resuspended in 20uL Ultrapure Endotoxin-free 0.1 M PBS (Thermo Fisher Scientific, MA, USA). The EV-containing supernatant was transferred to a new ultracentrifuge tube and spun at 100,000 ×g for 90 min at 4 °C. The supernatant was decanted, and EV pellets were resuspended via titration for 1 min in 20 μL Ultrapure Endotoxin-free 0.1 M PBS (Thermo Fisher Scientific, MA, USA) and stored in − 80 °C until quantification.

Immunolabeling for GSDMD and quantification was performed on optic nerve cross sections. Cryosections were incubated in 10% normal goat serum (I5256-10MG, Sigma-Aldrich) for 1 h at room temperature (RT) followed by incubation in GSDMD primary antibody (1:1000, rabbit monoclonal, ab209845, Abcam, United States) overnight at 4 °C. The following day, sections were washed in Ultrapure Endotoxin-free 0.1 M PBS (Thermo Fisher Scientific, MA, USA) and incubated in Alexa Fluor™ 488 secondary antibody (1:5000, Goat anti-Rabbit IgG, Invitrogen) for 2 h at RT. The sections were washed in 1 × PBS thrice (15 min per wash) and then stained (1:10,000 bisbenzimide, Sigma-Aldrich, MO, United States) to visualize the cellular layers, and cover slipped with aqueous mounting medium (Aquamount, cat. no. 18606; Polysciences, Warrington, PA). Immunofluorescence was viewed and captured with the A1 + confocal microscope at 20× and 40× magnification. Alexa Fluor™ 488 (detected in green channel) was pseudo-labelled as yellow LUT using ImageJ V2.0 software (NIH, MD, USA) for visualisation.