( for handling few cellsOn-chip CBNF s...)Flow cytometric analysis of human,,,primary cells using the Agilent 2100,,,bioanalyzer and on-chip staining

for handling few cells On-chip CBNF staining and steps for handling few cells are described in reference 2, using procedure B.
Experiments and Results
We have developed tools for measuring the transfection efficiency using a GFP reporter plasmid in primary cells on the 2100 bioanalyzer. Great emphasis was placed on being able to perform the analysis with a minimum of cells and to eliminate steps in the protocol where cells may be lost. These protocols facilitate working with very few cells. A concentrated cell buffer (2x CB) was added to the Cell Fluorescence Lab Chip kit. This buffer allows the exact adjustment of cell concentration and density of samples with few cells. Cells were transfected and stained on-chip with the live cell stain CBNF as described in reference 2. Only 30,000 cells were required for onchip staining of primary cells and subsequent measurement on the 2100 bioanalyzer. Figure 1 shows an optimization study conducted to reach best transfection efficiency in HUVEC cells. HUVEC cells were transfected with decreasing DNA:Lipofectamine (LP) ratios (1:1 to 1:10). Figure 1A shows the transfected samples as viewed through a fluorescence microscope 18 hours after transfection and figure 1B shows the corre-

sponding dot plots generated on the 2100 bioanalyzer. The selected region shows the positively transfected live cells. Figure 1C shows the GFP-histograms of the samples with the percentages of double positive cells. The recovery of live cells, as determined by manual counting under microscope after trypan blue straining, decreased as the amount of Lipofectamine was increased (compare listed percentages of recovery in figure 1A) due to the toxic effect of Lipofectamine on the cells. However, the transfection efficiency steadily increased up to a DNA:Li
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