(Rockford, IL). Fifty or 100 μL of the reconstituted standards or samples of the supernatant medium were isocitrate dehydrogenase inhibitor plated onto wells of plates coated with anti-human primary antibody and then incubated with 50 μL of a biotinylated detection antibody reagent at room temperature for 2 h. At the end of the incubation, the plate was washed three times and 100 μL of streptavidin–horseradish peroxidase solution was added to each well and incubated for 30 min at room temperature. Following another

three washes, 100 μL of tetramethylbenzidine substrate solution was added to each well and the colored product was allowed to develop at room temperature in the dark. After 30 min, 100 μL of stop solution was added and the absorbance of the samples was measured at 450 nm (Golub et al., 2008). As shown in Fig. 1, control wells were incubated with monocytes in serum-free conditioned media (SFCM) and stimulated (or not) by lipopolysaccharide. In the absence of doxycycline and lipopolysaccharide, <50 pg mL−1 of TNF-α was secreted by the monocytes, which was increased to 376.9 pg mL−1 of TNF-α when lipopolysaccharide was added to the culture. When doxycycline was added to the culture of the

lipopolysaccharide-stimulated monocytes in final concentrations of 0.1, 1 and 10 μM, the extracellular TNF-α levels were decreased by 46%, 52% and 71%, respectively. The effect of the same concentrations of doxycycline was much less dramatic on the production of IL-1β (Fig. 2). Monocytes secreted 58 pg mL−1 of IL-1β when lipopolysaccharide was added to the culture. However, when these cells were incubated in the presence of doxycycline at concentrations of 0.1, 1 and 10 μM, the extracellular IL-1β Ibrutinib datasheet levels were

only reduced by 9%, 16% and 16%, respectively. The extracellular levels of MMP-9, a major MMP secreted by monocytes, in the CM from lipopolysaccharide-stimulated monocyte cultures maintained in the presence of 0.1, 1 and 10 μM doxycycline, were initially analyzed by ELISA. Decreased MMP-9 levels were observed; 0.1, 1 and 10 μM doxycycline decreased MMP-9 levels by 18%, 20% and 41%, respectively (Fig. 3). In separate experiments, the monocytes were allowed to mature for 7 days into macrophages and the levels of both MMP-2 (72-kDa gelatinase) and MMP-9 (92-kDa gelatinase) Glycogen branching enzyme were assessed by gelatin zymography (Fig. 4). MMP-9 was consistently found to be more dominant than MMP-2 at days 1, 3 and 7, particularly at the later time periods; the MMP-9 levels progressively increased with the duration of the incubation, while MMP-2 remained constant. Moreover, doxycycline in final concentrations of 0–20 μM inhibited MMP-9 in a dose–response manner, but had no effect on MMP-2. A similar effect of these concentrations of doxycycline was observed when 0.1 μg mL−1 lipopolysaccharide was added to the macrophages in culture. Monocyte-derived macrophages were cultured with lipopolysaccharide in the presence of 0, 10 and 20 μM doxycyline for 2 days.