The background defines the lowest part of the range where the data is assessed. This allows debris to be excluded so that it can’t impact the ability to fit curves to the proliferation data. The loss factor represents the ratio between the peaks in terms of fluorescent intensity, which is not always perfectly a factor of 2.
The chi squared value in this context reflects the goodness-of-fit rather than defining whether a fit is acceptable or not. So, it is most useful to compare like samples to see if the nature of the curve fitting is comparable. You can also use the chi squared value to assess whether manual adjustment of the curve fitting is improving the fit or not.
The user can adjust the position and width of the GEN1 gate in order to influence the subsequent fitting of the curves. If you wish to adjust the position and the width of the gate itself, then select “Manual position” and “Manual width”, display the GEN 1 gate from the Gate List tab, and then adjust. The software will re-calculate the curves and statistics.
When other items, such as background and loss factor values, are set to “Manual”, the user can define them. In this case, the software will adjust within the defined constraints. When it is not manual, the software will adjust the specific values accordingly.
We recommend setting the Background to Manual (a value of 10 is often fine) but to let FlowLogic determine the loss factor and GEN 1 gate position/width itself. It is ok to leave the Number of Peaks at 12. This means FlowLogic will search all the data and won’t stop once it has detected the pre-defined number of peaks. The number of true peaks can be seen in the plot side drawer statistics window containing a reasonable number of cells.
From the Gate List tab, it is possible to display all of the gates that are created by the curve fitting algorithm. Only the size and position of the GEN 1 gate can be adjusted. All other gates will adjust with it, however. The statistics derived from these gates can be viewed in the File Inspector. However, be aware that the proliferation statistics are calculated based on the area under the automatically fitted curves.