Coralite488 conjugated anti mouse igg antibody

BMDMs were pretreated with KM for 1 h and then treated as mentioned above to activate the NLRP3 inflammasome. The supernatants were discarded, and the cells were lyzed with 0.5% Triton X-100 for 30 min. The lysates were centrifuged at 6,000 × g and 4°C for 15 min. The pellets were washed, resuspended in 500 μl of PBS and combined with 2 mM disuccinimidyl suberate (DSS, Sigma, St. Louis, MO, United States), and the samples were incubated at room temperature for 30 min with rotation and centrifuged at 6,000 × g and 4°C for 15 min. The cross-linked pellets were resuspended in 30 μl of sample buffer, boiled and then analyzed by immunoblotting.
For the ASC speck assay, BMDMs were stimulated to induce NLRP3 inflammasome activation, fixed with 4% PFA for 30 min and permeabilized with 0.5% Triton X-100 for 10 min. The BMDMs were incubated with anti-ASC antibody overnight at 4°C and then stained with CoraLite488-conjugated anti-mouse IgG antibody (1:100, Proteintech, Wuhan, China) for 1 h. The nuclei were stained with Hoechst 33342 for 15 min.

For the analysis of p65 nuclear translocation, BMDMs or PMA-differentiated THP-1 cells were incubated with KM for 1 h and then treated with LPS (300 ng/ml) for 1 h. The cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 30 min, and permeabilized with 0.5% Triton X-100 for 10 min. The cells were then blocked with 5% BSA for 1  h, incubated with anti-p65 antibody (1:100) overnight at 4°C, and stained with CoraLite488-conjugated anti-mouse IgG antibody (1:100, Proteintech, Wuhan, China) for 1 h. The cells were subsequently stained with Hoechst 33342 (Sigma, St. Louis, MO, United States) for 15 min. Images were captured with a Leica DMi8 fluorescence microscope.
For assays of NLRP3 and ASC colocalization, BMDMs or PMA-differentiated THP-1 cells on confocal dishes were fixed, permeabilized and blocked. The cells were stained with anti-NLRP3 and anti-ASC antibodies overnight and then with CoraLite594-conjugated anti-mouse IgG and CoraLite488-conjugated anti-rabbit IgG antibodies (1:100, Proteintech, Wuhan, China) for 1 h. The cells were incubated with Hoechst 33342 and imaged with a Leica DMi8 fluorescence microscope.