( pofectamine ratio of 1:8.When more L...)Flow cytometric analysis of human,,,primary cells using the Agilent 2100,,,bioanalyzer and on-chip staining

pofectamine ratio of 1:8. When more Lipofectamine was used transfection efficiency decreased again. This data shows that to obtain a maximum number of live transfected cells it is important to take the toxicity of the transfection reagent into account. For this experiment, a ratio of 1:6 was found to be optimal. In those cases where enough cells were obtained, samples were measured in parallel on a flow cytometer (figure 1D - only GFP histograms are shown). We also determined the optimal transfection condition for NHDF using a similar protocol. Figure 2 shows the results obtained when the cells were transfected with the optimal DNA:Lipofectamine ratio of 1:8. Figure 2 represents the dot plots of the control (A), the transfected sample (B) and the GFP histograms - control (C) and transfected (D). Although on average only 650 events were counted per sample data quality was very good for the histograms and dot plots. Figure 2E shows the untransfected and figure 2G the transfected samples viewed using a fluorescence microscope 18 hours after transfection. Figure 2F is a phase contrast photograph of the control cells.

Conclusion
Primary cells are valuable samples and are frequently limited in availability and lifespan. It can therefore be difficult to conduct experiments with these cells. The Agilent 2100 bioanalyzer with its low sample consumption together with an on-chip staining protocol proves to be a versatile tool for analysis of primary cells. The complete staining procedure takes place on chip and the analysis requires minimal amounts of cells and reagents, saving both time and reducing sample consumption.



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