Expression of mouse (top row) or rat (bottom row) MCP-1 by stimulated peritoneal cells. Mouse: Thioglycolate-elicited peritoneal macrophages from 6 month old BALB/c mice were stimulated with LPS (1 µg/ml; Sigma) for 5 hours in culture in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). Fc receptors were blocked using 0.5 µg of Fc Block™ (Cat. No. 553142). Cells were stained with 0.06 µg of FITC-rat anti-mouse Mac-1 antibody, fixed, permeabilized, and then stained with 0.25 µg of PE-hamster anti-mouse MCP-1 antibody (PE-2H5; Cat. No. 554443) by using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of PE-2H5 was blocked by the preincubation of the conjugated antibody with recombinant mouse MCP-1 (0.25 µg, Cat. No. 554590; middle panel), and by preincubation of the fixed/permeabilized cells with unlabeled 2H5 antibody (8 µg, Cat. No. 554711; right panel) prior to staining with the PE-2H5 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (middle panel) and unlabeled antibody blocking (right panel) specificity controls. Rat: Peritoneal cells from Lewis rats were harvested and plated in complete RPMI for one week. Cells were stimulated with LPS (1 µg/ml final concentration; Sigma) overnight in the presence of GolgiPlug™ (1 µg/ml; Cat. No. 555029), harvested and blocked for nonspecific staining with purified rat IgG. Cells were fixed, permeabilized, and then stained with 0.25 µg of PE-hamster anti-mouse MCP-1 antibody (PE-2H5; Cat. No. 554443). To demonstrate specificity of staining, the binding of PE-2H5 was blocked by the preincubation of the conjugated antibody with recombinant rat MCP-1 (0.25 µg, Cat. No. 555110; middle panel), and by preincubation of the fixed/ permeabilized cells with the unlabeled antibody blocking (right panel) specificity controls.