Immunocytochemistry analysis of THP-1 cells treated with 80nM TPA overnight then replaced with 100ng/mL LPS for 6 hours add 300ng/mL BFA for last 3 hours labeling IL-1 beta with Mouse anti-IL-1 beta antibody (HA601035) at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-IL-1 beta antibody (HA601035) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. beta Tubulin (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.

IL-1 beta Antibody (HA601035) in indirect ELISA. Indirect ELISA analysis of IL-1 beta was performed by coating wells of a 96-well plate with 50 µl per well of IL-1 beta antigen diluted in carbonate/bicarbonate buffer, at a concentration of 1 µg/mL overnight at 4℃. Wells of the plate were washed, blocked with StartingBlock blocking buffer, and incubated with 50 µl per well of a mouse IL-1 beta monoclonal antibody starting at a concentration of 20 µg/mL and serially diluting it to a concentration of 1.28 ng/mL for 2 hours at room temperature. The plate was washed and incubated with 50 µl per well of an HRP-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1:10,000 for one hour at room temperature. Detection was performed using an Ultra TMB Substrate for 5 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.