Reproducibility and sensitivity are imperative to any analytical method used for critical decision making. AsedaSciences® has extensively characterized the performance of our screens and, in the process, developed proprietary tools that ensure the quality of our results. Following is a snapshot of the performance and the types of biological fingerprints our SYSTEMETRIC® cell physiology screen can generate to aid with critical decisions earlier in drug discovery.
• MITOCHONDRIAL MEMBRANE POTENTIAL (MMP) •
The above plots show the effect of 1 uM FCCP and 1 uM Amiodarone in one of our cell physiology screens in comparison to the negative control (contains no compound) for both MMP and Cell Cycle. The square box labeled as “normal” has been maintained across all plots to enable a visual as to the extent of impact on MMP for these low compound concentrations. As the fluorescence increases, this indicates a decrease in MMP. You will notice the difference in response between the two compounds, with FCCP causing a decrease in MMP for 90% of the cells, while Amiodarone shows two distinct populations, with only 30% of cells showing a decrease in MMP, with the S + G2/M phase exhibiting the greatest change.
Here, we demonstrate different, but expected, profiles for two separate compounds for MMP alone; again, showing the contribution of this parameter to the overall multi-parametric fingerprint. The sensitivity of the method can be demonstrated using Valinomycin, which disrupts MMP at the lowest concentration (0.005 uM). Terfenadine, which is known to disrupt MMP at high concentrations, is provided for comparison.
• IMPORTANCE OF MEASURING POPULATION HETEROGENEITY •
Flow Cytometry makes it possible to detect, quantify and visualize cell population heterogeneity, such as sub-populations and rare events, as demonstrated with MMP. Here, we provide some further examples using light scatter (as an indicator of morphological changes), Reactive Oxygen Species (ROS), Glutathione and Cytoplasmic Membrane Integrity. The compound used is 1.0 uM FCCP.
Through simultaneous measurement of additional parameters, we can add to the knowledge stream of the phenotypic fingerprint. Flow cytometric analysis of cells in a single well treated with 1 uM FCCP reveal that while the majority (~80%) of the cells show normal cell light scatter characteristics (green events, left panel), indicating normal morphology, a sub-population of ~50% of these cells show an increase in ROS (exhibited by an increase in fluorescence in the right panel). The right panel is a four-dimensional “radar plot” showing the expression of ROS, orthogonal light scatter, Cell Membrane Integrity and Glutathione in one plot.
Therefore, if we look at the phenotypic fingerprints of 1 uM FCCP, we detect very unique indicators within the heterogeneity of cellular effects. This can only be effectively demonstrated by analyzing a large number of cells (in this case, 10,000). This shows our ability to generate large data sets of compound effects in a very sensitive system. We have exhibited that you can see normal light scatter (morphology) in 80% of cells at this concentration of FCCP; however, 50% of these are showing an increase in ROS, likely caused by the decrease in MMP (cell cycle related) in approximately 90% of the cells. When combined with the other concentrations of the dose response, this type of profile provides a content rich biological fingerprint that can be used for comparison against other compounds. Thus, our proprietary algorithm can be used for a similarity analysis to determine which compounds fall into the category of “good, bad or ugly.”
• SYSTEMETRIC BIOLOGICAL FINGERPRINTS •
Finally, to provide some additional “variety” to the types of phenotypic fingerprints created by different compounds, below are three different examples in comparison to the negative control in a four-dimensional radar plot of ROS, Glutathione, Cellular Membrane Integrity, and Side Light Scatter.