( f of the cells werestained with SYTO...)Apoptosis detection by annexin V and,,,active caspase-3 with the Agilent 2100,,,bioanalyzer

f of the cells were stained with SYTO16 (an intercalating nucleic acid stain), washed again and treated with a permeabilization/ fixation solution. They were subsequently stained with anti active caspase-3 antibodies and a Cy5- conjugated secondary antibody (figure 4 A). The other half of the treated cells were stained with calcein/annexin V-Cy5 as described above (figure 4 B). Both sample groups were measured on the 2100 bioanalyzer and on a conventional flow cytometer. Only 20,000 cells of each prepared cell sample were loaded onto the chip (500-1000 cells analyzed per sample), whereas 100,000 cells were required for measurement on the flow cytometer (10,000 cells analyzed per sample). As expected, the percentages of live apoptotic cells are similar for both assays. The Agilent 2100 bioanalyzer displayed good reproducibility and yielded data comparable to the flow cytometer system.

Materials and methods
Cells were cultured in RPMI medium containing 10 % fetal calf serum, penicillin/streptomycine, 1 mM Sodium Pyruvate and 2 mM Lglutamine.
a) Induction of apoptosis and annexin V assay
Reagents

Camptothecin: Prepare a 10 mM solution in DMSO.
Annexin V-Biotin Apoptosis Detection Kit: Includes annexin V-biotin, 5x binding buffer and media binding reagent
Fluorolink Cy5 labeled streptavidin: Reconstitute with 1 ml distilled water to yield a concentration of 1 mg/ml
Paraformaldehyde 16 % solution EM Grade (optional)
Calcein-AM: Dilute the original stock with DMSO to yield a 500 M solution.
Protocol for annexin V assay
1. Treat Jurkat cells at a concentration of 5 x 105/ml with 1 M camptothecin or 0.5
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