Immunocytochemistry analysis of IL-1β expression on stimulated PBMC (left figure). PBMC were isolated from human peripheral blood by density gradient centrifugation and were cultured for 6hr at 37°C with human IFN-γ (20 ng/ml, Cat. No. 554616). The cells were subsequently stimulated with 1 μg/ml LPS (Sigma No. L-8274) and were incubated with GolgiStop™ (Cat.No. 554724) overnight at 37°C. The activated cells were harvested and the level of IL-1β producing cells was detected by immunocytochemistry using a three-step staining procedure that employs a Biotin Goat Anti-Mouse IgG (Cat. No. 559286) secondary antibody and Streptavidin HRP (Cat. No. 550946). To demonstrate specificity of staining the binding of the Purified Mouse Anti-Human IL-1β (Cat. No. 550007) antibody was blocked by the preincubation of the purified antibody with excess recombinant human IL-1β protein (Cat. No. 554602; data not shown). (Nomarski optics, original magnification 400 X). Flow cytometric analysis of IL-1β expression on human PBMC (right figure). PBMC were cultured with recombinant human IFN-γ (10 ng/ml, 2h, 37°C),then stimulated with LPS (1 µg/ml) and GolgiStop (2 µM,) overnight at 37°C. Cells were harvested, washed, fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (Cat. No. 554722), then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with Purified Mouse Anti-Human IL-1β (Cat. No. 550007; solid line histogram) or Purified Mouse IgG1 κ Isotype Control (Cat. No. 55414). Secondary staining was carried out with FITC Goat Anti-Mouse IgG/IgM (Cat. No. 555988). Fluorescence histograms depicting IL-1β (or Ig istoype) expression were derived from gated events with the side and forward light-scatter characteristics of viable monocytes.