Figure 1.
Example test plate designed to be processed in Cheburator.
The sample plate with 1 control row (H), 1 row for the reference drug (A), and 4 rows for 4 novel studied compounds (B, C, D, and E, respectively) tested in 4 concentrations. Please note that not all rows have to be used on the plate, and different ranges of concentrations for different compounds can be applied in the test. Concentrations are highlighted in red. Control of untreated cells (CC) and control of media (CM) are in blue and green, respectively.
Figure 2.
Graphical explanation of data analysis algorithms used in Cheburator.
(A) Two types of IC50 which can be estimated by the software. Absolute IC50 is defined as the concentration of tested compound which results in 50% inhibition of cell growth as defined by assay controls (pink lines). Relative IC50 is defined as the concentration of tested compound which results in half of the maximum inhibition of cell growth attainable for that particular compound (orange lines). Imax is defined as upper plateau of the dose-response curve (solid orange line). (B) Example of a dose-response curve drawn on the basis of 4 values (black circles). The simple two-point method uses 2 data points bracketing 50% inhibition of proliferation (green lines) to estimate the IC50. In the second method, a linear model is built with the intercept and the slope, which are calculated by linear regression analysis using data points which are >0% and <100% (red lines). These parameters are later used to estimate IC50. In the third method, the software uses all data points to build nonlinear regression model and estimate IC50 (blue lines). Absolute IC50 values are shown on this plot, but software can also estimate relative IC50 in linear and nonlinear regression analysis.
Figure 3.
The built-in converter for.csv files.
The line containing the data from the first row of the plate is selected in the picture. Other rows have to follow immediately after the first row. All rows before and after the plate data will be discarded. Each row is then divided into separate values using the delimiter specified in the combo box below (semicolon is the default, but other delimiters are also supported). Since each row could contain more values than the number of wells, the user also has to specify in the edit box below which value in the row will be treated as the optical density value for the first well. Data for the subsequent wells have to follow immediately after, separated by delimiters. Note that the user can use a period or comma as the decimal separator for data files. Cheburator will automatically convert all decimal separators into periods, which are the default.
Figure 4.
Main window of the program, showing the test parameters and plate data table.
The absorbance values in wells are highlighted with the intensities proportional to the ratio between their respective values and control values.
Figure 5.
This window allows the user to change or discard particular values from the analysis. Any changes will be noted in the final report.
Figure 6.
Marking of the discarded and changed values.
Changed values appear in red, and discarded values are crossed out after data modification.
Figure 7.
The Results tab after the analysis.
It is showing the dose range applied, mean and standard deviations of absorbance values for every triplicate, calculated respective percentages of inhibition of cell proliferation, estimated IC50 values, its type, method of calculation, and 95% confidence intervals (for regression analysis).
Figure 8.
The Graphs tab after the analysis.
It is showing the dose-response curves, individual values of percentages of inhibition for each data point, method of calculation used, IC50 values, its type, 95% confidence intervals, and goodness of fit (for regression analysis).
Figure 9.
Example of test report printed by Cheburator.
This sheet contains the original file’s name, date and time, the name and date of the test, the calculated IC50 values, its type, 95% confidence intervals, and goodness of fit (for regression analysis), the percentages of growth inhibition for every concentration and compound, the dose-response curves, the mean optical densities and their standard deviations, the test control values, and the type of the method used for calculation. A table with raw plate data is printed at the bottom part of the report.
Figure 10.
This tab allows user to customize different analysis and report parameters of the software.