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neo and Bcl-2 cells were set up and treated with staurosporine as described in (17). After centrifugation at 600g for 10 min at 4°C, the cell pellets were resuspended in PBS containing 5 μM rhodamine 123 (Sigma) and incubated at 37°C for 15 min. The cell suspension was centrifuged in a microcentrifuge for 30 s, and the pellet was resuspended in 20 μl of PBS, plated onto a 25-mm round glass cover slip coated with poly-D-lysine, and mounted into a perfusion chamber for confocal imaging. The fluorescence images were collected with a Meridian Insight-Point Laser scanning confocal microscope (Meridian Instrument) equipped with a Zeiss Axioplan microscope. The objective lens was a 100x numerical aperture 1.4 PlanApo lens. The aperture size of the pinhole was 10 to 40 μm. Confocal optical sections were estimated to be less than 1 μm in thickness. Cells were excited with the 488-nm line of an argon laser, and emitted fluorescence was detected through a 530/30 band-pass filter with an intensified, cooled charge-coupled-device camera. A typical cell from a population of ̃ 100 was presented.
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note
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We thank R. Jemmerson of University of Minnesota for monoclonal antibody to cytochrome c; S, McKnight, J. L. Goldstein, and M. S, Brown for critically reading the manuscript; and T. Greenamyer for help with confocal microscopy. Supported by the start-up fund from Emory University, an American Cancer Society research grant, and a Junior Faculty award (to X.W.).
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