Tmb chromogen solution

96-well EIA/RIA plates (Corning) were coated overnight at 4 °C with 20 μg of G protein per plate in coating buffer (Solarbio). All wells were blocked with 100 µl of blocking buffer (5% skim milk in PBS) for 1.5 h at 37 °C. After standard washes and blocks, serum (2x serially diluted, starting at 100x dilution) in 5% skim milk in PBS was incubated at 37 °C for 2 h. Plates were washed four times in PBS with 0.1% Tween 20 (PBST) before addition of HRP-conjugated goat anti-mouse IgG (Abcam, Cat No. ab6789, diluted 1:20,000), IgA (Abcam, Cat No. ab97235, diluted 1:10,000) or HRP-conjugated goat anti- hamster IgG (Abcam, Cat No. ab6892, diluted 1:15,000) diluted in 5% milk in PBS for 1.5 h at 37 °C. Plates were washed as before prior to being developed with 100 µl/well of TMB chromogen solution (Beyotime) for 15 min. Substrate reactions were stopped by the addition of 50 µl/well of stop solution for TMB Substrate (Beyotime) before reading plate absorbance at 450 nm (OD450). The cutoff value was defined as 2.1-fold of OD450 values from the sample of nonvaccinated mice. The reciprocal of the maximum sample dilution with OD450 values equal to or greater than the cutoff value was used to calculate the endpoint binding antibody titers.

Dimethyl sulfoxide (DMSO) (D8371, Solaibao, Beijing, China); Cell Counting Kit-8 (CA1210, Solaibao); BCA Protein Concentration Determination Kit (P0012S, Beyotime, Shanghai, China); RIPA buffer (R0010, Solaibao); Hematoxylin and Eosin Staining Kit (C0105S, Beyotime); 4% paraformaldehyde (BL539A, Biosharp, Hefei, China); 10% neutral buffer formalin fixative (Thermo right, Changchun, China); TMB Chromogen Solution (P0211, Beyotime); DAPI (C0065, Solaibao); Anti-GSDMD antibodies (ab219800, Abcam, Cambridge, UK); Anti-NLRP3 antibodies (ab214185, Abcam); Anti-caspase-1 antibodies (ab138483, Abcam); β-actin polyclonal antibody (20,536-1-AP, Proteintech, Rosemont, IL, USA); Anti-caspase-1 antibody (AB1871, Sigma,-Aldrich, USA); NLRP3 recombinant rabbit monoclonal antibody (sc06-23, Invitrogen, Waltham, MA, USA); Cy3-labeled goat anti-rabbit IgG (H + L) (A0516, Beyotime); Goat-anti-rabbit IgG (H + L) (A0208, Beyotime); Biotin-labeled Goat Anti-Rabbit IgG(H + L) (A0277, Beyotime); Empagliflozin (HY-15409, MedChemExpress, Princeton, NJ, USA); ECL Kit (Millipore, Billerica, MA, USA). CO₂ constant temperature incubator (Thermo Fisher, USA); fluorescence microscope (DM500, Leica, Germany); Electrophoresis instrument electrophoresis system (Bio-Rad, Hercules, CA, USA); ChemiDoc™MP system (Bio-Rad).

Flag antibody (M185-3L, MBL) was diluted with antibody coating buffer [carbonate buffer (pH 9.6)] to 1 µg/ml to cover the 96-well plate at 4°C overnight. The coated plate was washed with PBST (PBS supplied with 1% Tween-20) to remove the unbound antibody. Purified inactive mR3 proteins were firstly diluted with storage buffer [50 mM Tris-HCl (pH 8.0), 300 mM NaCl] (Glow et al., 2015 (link)), followed by dilution with PBST to reaction concentration (0.8 µM for monomer proteins, 0.4 µM for dimer proteins). The diluted proteins were transferred to an antibody-coated plate to incubate for 2 h at room temperature, followed by washing with PBST. Biotin-labeled dsRNAs were diluted with LSB [10 mM Tris-HCl (pH 7.5), 5 mM NaCl, 1 mM MgCl₂, 0.1 mg/ml bovine serum albumin] or HSB [20 mM HEPES-KOH (pH 7.5), 140 mM KCl, 12 mM NaCl, 2 mM MgCl₂, 5% glycerol] to 0.02 µM to bind with coated inactive mR3 proteins for 1 h at 37°C, followed by washing with PBST and incubating with Streptavidin-HRP antibody (Abcam, ab7403, 1:1000) at 4°C overnight. After incubation and washing, the plate was stained with TMB Chromogen Solution (Beyotime, P0209) at 28.5°C. After sufficient color development, the staining reaction was stopped with 2 M H₂SO₄. The staining result was read out by microplate reader (EnSpire, PerkinElmer) at 450 nm.