Alexa fluor 488

ARIAD ligand (AL), D/D Solubilizer from Takara, ref: 635054.
Anti-GFP from Sigma Aldrich (ref: 11814460001), anti-Ds-Red from Ozyme (ref: 632496), anti-myc from Merck Millipore (ref: 6549), anti-4.1N from BD Biosciences (ref: 611836) anti-SAP97 from NeuroMab (ref:75–030), anti-GluA1 from NeuroMab (ref: 75–327). Secondary antibodies from Molecular Probes: goat anti-mouse Alexa Fluor-568 (ref: A11004), goat anti-rabbit Alexa Fluor-488 (ref: A11008); from Li-Cor: goat anti-mouse (ref: 926–32210), goat anti-rabbit (ref: 926–68021).

Cells (5000 cells/250 µl media) were seeded on 8-chambered slides and cellular attachment was allowed overnight. The cells in the chamber slides were washed 3 times in PBS and fixed with 4% paraformaldehyde for 10 min at room temperature and blocked with 1% BSA in PBS for 30 min. The primary antibody was applied for 1 h at room temperature, and then washed 3 times with PBS for 30 min. The slides were then incubated with the second antibody conjugated with Alexa Fluor 594 or Alexa Fluor 488 (LI-COR Biotechnology, Lincoln, NE, USA) for 1 h at room temperature. Finally, the slides were washed 3 times with PBS, mounted with mounting medium containing DAPI (Vector laboratories, Burlingame CA, USA) and observed with a fluorescence microscope (Nikon A1R laser scanning confocal imaging).

Lipopolysaccharide was obtained from Sigma (St Louis, MO, USA). IRF3 agonist, KIN1148 and ROS scavenger, N-acetyl-l-cysteine (Nac) were purchased from Medchem Express (Shanghai, China). IRF3 siRNA, STING siRNA and the scrambled siRNA were acquired from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies against NLRP3, STING, phospho-IRF3 (P-IRF3), total-IRF3 (T-IRF3), CD45, CD68, IL-18, BCL-2, BAX, TXNIP, ASC and Trx were purchased from Abcam (Cambridge, UK). Primary antibodies for Caspase1 and IL-1β were purchased from Proteintech Group (Rosemont, USA). Primary antibodies against proliferating cell nuclear antigen (PCNA), GAPDH, C-Casepase3 and Caspase3 were acquired from Santa Cruz Biotechnology (Dallas, TX, USA). Pam3Cys-Ser-(Lys)4 was purchased from Abcam (Cambridge, UK). The GTVision™+ Detection System/Mo&Rb reagent was obtained from Gene Technology (Shanghai, China). The Alexa Fluor 488- and 568-goat anti-rabbit secondary antibodies used in immunofluorescence staining were purchased from LI-COR Biosciences (Lincoln, USA). Lactate Dehydrogenase Assay Kit (Colorimetric) and Cell Counting Kit 8 (WST-8/CCK8) were obtained from Abcam (Cambridge, UK). Other chemicals in this study were of analytical grade.