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Protokoll
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A. Staining Procedure: 1. Induce apoptosis in cells (1 x 10 6 /ml) by desired method. Concurrently incubate a control culture without induction. An additional control can be prepared by adding the caspase family inhibitor Z-VAD-FMK at 1 μ l/ml to an induced culture to inhibit caspase activation. 2. Aliquot 300 μ l each of the induced and control cultures into eppendorf tubes. 3. Add 1 μ l of FITC-VAD-FMK into each tube and incubate for 0.5-1 hour at 37 o C incubator with 5% CO 2 . 4. Centrifuge cells at 3000 rpm for 5 minutes and remove supernatant. 5. Resuspend cells in 0.5 ml of Wash Buffer, and centrifuge again. 6. Repeat Step 5. Proceed to B, C, or D depending on methods of analysis. B. Quantification by Flow Cytometry: For flow cytometric analysis, resuspend cells in 300 μ l of Wash buffer. Put samples on ice. Analyzing samples by flow cytometry using the FL-1 channel. C. Detection by Fluorescence Microscopy: For fluorescence microscopic analysis, resuspend cells in 100 μ l Wash buffer. Put one drop of the cell suspension onto a microslide and cover with a coverslip. Observe cells under a fluorescence microscope using FITC filter. Caspase positive cells appear to have brighter green signals, whereas caspase negative control cells show much weaker signal. D. Analysis by Fluorescence Plate Reader: For analysis with fluorescence plate reader, resuspend cells in 100 μ l 1X Wash Buffer and then transfer the cell suspension to each well in a black microtiter plate. Measure the fluorescence intensity at Ex. = 485 nm and Em. = 535 nm. For control, use wells containing unlabeled cells.
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