Annexin 5 fitc

When 50–60% confluent in T25 flasks, both LAPC-4-AI and PC-3 cells were treated with vehicle control, 40μM EGCG+5μM Q, 5nM Doc, or EGCG+Q+Doc for 48h. Cells were trypsinized and monolayers attaching to the bottom were collected. The procedures for cell cycle and apoptosis analysis using a small cytometer Cellometer Vision (Nexcelom Bioscience LLC, Lawrence, MA) were described previously [29 (link), 30 (link)] with minor modifications. Briefly, for cell cycle assay cells were centrifuged and pellet resuspended and fixed in cold methanol. The cells were centrifuged again and pellet was stained in propidium iodide (PI) solution (Nexcelom Bioscience LLC) for imaging cytometry using Cellometer Vision. For apoptosis assay, cells were centrifuged and pellets were resuspended in Annexin V binding buffer and double-stained with Annexin V-FITC (Nexcelom Bioscience LLC) and PI (Nexcelom Bioscience LLC) for Cellometer analysis. A positive control was generated by heating cells in a 45°C water bath for 10min. Non-treated cells were used as negative control. Both controls were processed with the samples. The fluorescence data generated by the Cellometer software were converted into FCS files and analyzed by De Novo FCS Express 4 software (Los Angeles, CA). The experiment was performed in triplicate.