Fitc conjugated annexin 5

The surface antigens on monocytes and the induction of apoptosis were analyzed by flow cytometry. Briefly, after the incubation for 48 h, the cells were washed once by phosphate-buffered saline (PBS) containing 2% normal human serum and 0.1% sodium azide (staining buffer). The cells were then reacted in suspension with saturating concentrations of FITC-conjugated mAbs for 30 min at 4 °C. After the cells were washed once with staining buffer and then twice with PBS, they were counterstained with phycoerythrin (PE)-conjugated annexin V (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. The cells were then analyzed using Cell Lab Quanta SC (Beckman Coulter, Miami, FL). To identify viable cells, the gating for the staining with annexin V was used. In some experiments, the cells were stained with FITC-conjugated annexin V (R&D Systems) and propidium iodide (PI) and were analyzed by flow cytometry.

MDM and THP-1 macrophages were grown in 24-well plates (5 × 10⁵ cells/well). Cells were exposed to organic extracts for 24 h at the following concentrations: 1, 5, and 10 μg/ml. Next, cells were washed twice with complete RPMI-1640 medium, and MitoTracker Red CM-H₂XROS (500 nM) was added, and the incubation was continued for 40 min in the dark. Cells were washed twice with PBS and then analyzed with FlowJo (Tree Star, Inc., Ashland, OR). Typically, 100,000 events were acquired. Unexposed cells treated for 4 h with 200 ng/ml staurosporine (Sigma-Aldrich) were included as a positive apoptosis control. Apoptotic changes were identified by staining macrophages with FITC-conjugated annexin V according to the manufacturer instructions (R&D Systems). Specific binding of annexin V was achieved by incubating 10⁶ macrophages in 60 μl of binding buffer with annexin V for 15 min at 4°C. To differentiate between early apoptosis and necrosis, macrophages were simultaneously stained with propidium iodide (PI) before analysis. The binding of annexin V and PI to the cells was also measured by flow cytometry (FACSAria II; BD Biosciences). At least 10,000 cells were counted in each sample and a gate based on forward and side scatters was set to exclude cell debris.