Annexin 5 binding detection kit

Cells (~3 x 10⁵) were plated in 60mm diameter tissue culture plates in RPMI medium and allowed to grow for 48 hours. Then the spent culture medium was replaced with fresh 2ml DMEM medium and the cells were treated with MK591 (40μM) or ibuprofen (40μM) for 24 hours at 37°C. Then the cells in the plate were treated with FITC-labeled annexin-V and propidium-iodide (PI) for 15 minutes in the dark using Annexin V-Binding Detection Kit following a protocol supplied by the manufacturer (BD Biosciences). After washing, cells were photographed with a Nikon digital camera attached to a LEICA fluorescence microscope at 20X. Images were acquired by a Nikon digital camera attached to a Zeiss fluorescence microscope and the data were processed with a Dell computer using Q-Capture-7 Pro software.

Cells (~3 × 10⁵) were plated in complete medium and allowed to grow for 48 hours. On the day of experiment, the spent culture medium was replaced with fresh 2ml medium and the cells were treated with MK591 or ibuprofen for 24 hours at 37°C. Then the cells were treated with FITC-labeled annexin-V and propidium-iodide for 15 minutes in the dark using an Annexin V-Binding Detection Kit following a protocol supplied by the manufacturer (BD Biosciences). After washing, cells were photographed using a Nikon digital camera attached to a LEICA fluorescence-microscope at 20×. Image acquisition and data processing were done with a Dell computer attached to the microscope using Q-Capture 7 software.

C4-2B prostate cancer cells (~3×10⁵) were plated in RPMI medium and allowed to grow for 48 h. The spent culture medium was replaced with fresh 2 ml RPMI medium and the cells were treated with MK591 or ibuprofen for 24 h at 37°C. Then the cells in the plate were treated with FITC-labeled annexin-V and propidium-iodide (PI) for 15min in the dark following a protocol supplied by the manufacturer with the Annexin V-Binding Detection Kit (BD Biosciences). After washing, cells were photographed with a Nikon digital camera attached to a Leica fluorescence microscope at x200. Image acquisition and data processing were done with a Dell computer attached to the microscope using Q-Capture Pro7 software.