Introduction
Accurate analysis of data is critical to ensuring integrity of experimental results1. Consistent data evaluation methods reduce the opportunity for mis-analysis and process-related errors2. The SoftMax® Pro 6.4.1 Import Feature facilitates the systematic application of statistical and mathematical methods by allowing the user to import raw data in microplate format acquired from any scientific instrument into a SoftMax Pro protocol. In addition, data can be imported into validated, pre-written protocols using SoftMax Pro GxP compliance software for GLP and GMP laboratories.
Import Feature
The FLIPR® Tetra High Throughput Cellular Screening System from Molecular Devices identifies early leads against GPCR and ion channel receptors. Assay data is acquired and analyzed by the ScreenWorks® System Control Software, which is exclusive to FLIPR systems. Using the SoftMax Pro 6.4.1 Import Feature, data from an experiment using a Molecular Devices FLIPR® Potassium 6 Assay Kit was imported into SoftMax Pro 6.4.1 for analysis (Figure 1).
Figure 1. SoftMax Pro 6.4.1 import workflow.
Materials and methods
Chinese Hamster Ovary (CHO) cells expressing stable human Kv11.1 channel, provided by ChanTest Corporation (Cleveland, OH), were treated with the FLIPR Potassium Assay Kit according to the manufacturer’s directions. Varying concentrations of hERG inhibitors, Sigma Aldrich (St. Louis, MO), were added to the cells during detection on a FLIPR Tetra System with ICCD camera. FLIPR-generated kinetic data was exported as a .seq1 file and opened in Microsoft Excel. The raw data was arranged into plate format using the provided SoftMax Pro import template, saved as a tab-delimited text file, and imported into a pre-written SoftMax Pro protocol (Figure 2). Upon data import, SoftMax Pro automatically assigned inhibitor concentrations, performed maximum minus minimum relative fluorescence unit (RFU) data reduction, and calculated the standard deviation, %CV, and Z factor. Custom conditional formulas were set up to automatically remove outlier points from further calculations and display the EC50 values in a custom notes section for easy report viewing. In addition, the RFU values (max-min) for each compound were automatically plotted on the same graph using a 4-parameter curve fit to determine the EC50 values (Figure 3).
Figure 2. SoftMax Pro 6.4.1 Import Feature. Located in the main menu, the Import Feature allows for quick import of data using the SoftMax Pro import template.
Figure 3. EC50 estimates from multiple dose-response experiments in SoftMax Pro 6.4.1 Software. CHO-hERG cells expressing stable human Kv11.1 channel, provided by ChanTest Corporation (Cleveland, OH), were treated with varying concentrations of cisapride, dofetilide, flunarizine, haloperidol, quinidine, and terfenadine over time using the FLIPR Potassium Assay Kit. The resulting kinetic data from ScreenWorks was imported into SoftMax Pro 6.4.1 for analysis. The mean RFU value (max-min) for each concentration was plotted using a 4-parameter curve fit to determine the EC50 of each compound.
Conclusion
The SoftMax Pro 6.4.1 Import Feature enables import of previously incompatible data formats for analysis and interpretation using one software system. As shown in Figure 3, high throughput data acquired on the FLIPR Tetra System was successfully imported into SoftMax Pro 6.4.1 to determine the EC50 values of several hERG inhibitors using CHO cells stably transfected with human Kv11.1 channel
The powerful features of SoftMax Pro 6 allowed for a more complete analysis of the imported FLIPR Tetra System data. Imported plate data was cloned to test different kinetic reduction options within the same experiment protocol. The curve overlay feature simplified the data report and facilitated curve comparison between the 19 different curve fit options offered in SoftMax Pro 6. Finally, the EC50 values for each compound were pulled into a custom notes section along with the final graph to provide a summary of the experimental results (Figure 4).
Figure 4. The notes section can be customized to display images, graphs, and data results
To ensure accuracy of data analysis, the National Academy of Sciences recommends providing scientists with appropriate tools for management of research data3 . The SoftMax Pro 6.4.1 Import Feature is a tool that streamlines and consolidates data reduction from scientific instruments of diverse manufacturers. Furthermore, importing microplate or cuvette data into SoftMax Pro GxP compliance software for analysis reduces time and money spent on validating additional software platforms and protocol calculations in GLP and GMP environments.
References
1. Shephard RJ. Ethics in exercise science research. Sports Med 2002; 32(3):169–183
2. Pascal CB. Managing Data for Integrity: Policies and Procedures for Ensuring the Accuracy and Quality of the Data in the Laboratory. Science and Engineering Ethics 2006 Jan; 12(1):23-39.
3. National Academy of Sciences. Committee on Ensuring the Utility and Integrity of Research Data in a Digital Age; Committee on Science, Engineering, and Public Policy (COSEPUP); Policy and Global Affairs (PGA); Institute of Medicine (IOM): Ensuring the Integrity, Accessibility, and Stewardship of Research Data in the Digital Age. Washington, D.C: The National Academies Press; 2009.