( .Detection of antibody-stained cells...)Detection of antibody-stained cell,,,surface and intracellular protein targets,,,with the Agilent 2100 bioanalyzer

. Detection of antibody-stained 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. 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 the intuitive software package supplied with the Cell Assay Extension with no requirement to manually set instrument- specific parameters.
Results
a) Extracellular CD3 staining of Jurkat cells

Jurkat cells, a cell line derived from an acute human T-cell leukemia, were harvested and washed. After staining with calcein, the cells were incubated with APC-labeled anti hCD3-antibody. After washing and resuspending the cells in cell buffer the samples were loaded onto the chip and measured in the Agilent 2100 bioanalyzer. Figure 1 shows representative histograms of a mixture of calcein-only and calcein and antibody treated cells. The data obtained with the 2100 bioanalyzer are of comparable quality as data generated with the same samples on a conventional flow cytometer. Figure 2 shows the reproducibility of the measurement over several chips and compares the data obtained from the Agilent 2100 bioanalyzer with flow cytometer measurements.

b) Extracellular CD4 staining of CCRF-CEM cells

CCRF-CEM cells, a cell line derived from an acute human lymphoblastic leukemia, were harvested and washed. After staining with calcein, the cells were incubated with APC-labeled anti hCD4-antibody. After washing and resuspending the cells in cell buffer
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