click to enlarge Figure 1: InCyte software calculated staurosporine EC50 in 7-AAD- and caspase-9-SR-labeled HeLa cells. Fluorescence data were acquired on a guava easyCyte 8HT flow cytometer. (Source: Millipore)

New automated analysis software for benchtop flow cytometry systems allows researchers to conduct multiparametric cell analysis, regardless of their level of expertise or access to a core facility. Traditionally, flow cytometry users have manually set markers, or “gates,” between cell populations using simple analysis tools. However, when measuring multiple protein targets, new software packages provide the required statistical power and complex modeling of multi-dimensional assay data.¹
 
This new software allows researchers to combine groups of data using statistically calculated gating to correlate cell populations and to construct heat maps or EC50 curves. Because results are compared at the experiment level, rather than on an individual sample basis, such software can further the understanding of interactions between different biological processes and the results of high-throughput, cell-based assays.

InCyte flow cytometry analysis software, available with the guava easyCyte flow cytometry systems, brings this kind of cell analysis to the benchtop. In the example, HeLa cells were titrated with various compounds and then assayed for apoptotic signaling (caspase-9 activity) as well as cell death phenotype (nuclear envelope breakdown).

The resulting four-dimensional data were mined using InCyte software to determine the concentration of staurosporine required to cause 50% of cells to show apoptotic signaling and nuclear envelope breakdown. Forward and side scatter (Figure 1A) were used to discriminate cells from debris. Cells positive for both caspase-9 activity and nuclear membrane breakdown were identified with a second gate (Figure 1B). Grouping data from selected treatments (Figure 1D), the software displayed direct count of number of cells in each well that fell within the selected cell population (Figure 1C).  Finally, using the EC50 template, the data were fit to a dose response curve (Figure 1E) from which the staurosporine EC50 was calculated.

The software improves analysis by eliminating user-to-user gating variation, and integrates acquisition to enable real-time plot adjustments. When combined with benchtop systems and assay reagent kits, automated analysis software gives researchers the opportunity to apply flow cytometry to their particular research question, even without flow cytometry experience or expert guidance.

Reference
1. Pyne S, et al. Automated high-dimensional flow cytometric data analysis. PNAS. 2009;106(21):8519.