( taining with anti-active caspase-3an...)Apoptosis detection by annexin V and,,,active caspase-3 with the Agilent 2100,,,bioanalyzer

taining with anti-active caspase-3 antibodies. Following fixation, permeabilization and staining, the activity of caspase-3 can be measured by flow cytometry or fluorescence microscopy techniques.
Here, we describe the measurement of percentages of apoptotic cells within cell populations using the Agilent 2100 bioanalyzer. The apoptotic process was induced in Jurkat cells, a cell line derived from an acute human T-cell leukemia, either by treatment with camptothecin or by incubation with anti Fas-antibodies. Different readouts were chosen either annexin-presenting cells within the live cell population were detected or the percentage of cells with active caspase-3 was measured in the total cell population.
Experimental
The Agilent 2100 bioanalyzer and Cell Assay Extension were obtained from Agilent Technologies Deutschland GmbH (Waldbronn, Germany). Detection of apoptotic cells was performed on the Agilent 2100 bioanalyzer in combination with the Cell Fluorescence LabChip Kit and the Cell Fluorescence software. The kit includes 25 chips and reagents required to perform the analysis of pre-stained cells. Stained cell samples were resuspended in an isobuoyant cell buffer at 2 x 106 cells/ml and loaded onto the chips as described in the reagent kit guide. Data acquisition was performed using an intuitive software package with no requirement to manually set instrument specific parameters.
Results
a) Induction of apoptosis with camptothecin; annexin V staining
In a first application example, Jurkat cells were induced with camptothecin and harvested after 0, 4 and 16 hours of treatment. After washing, the cells were incubated with annexin V-biotin. Cells were washed again and stained


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