Influx Index Sorting Data Files
Contents
Influx Index Sorting Data Files contain fluorescence information on both the sorted and unsorted events. To isolate the sorted events:
1. Insert a plate heat map for the index sort data file.
2. Select the parameter of interest on the y-axis (plate heat maps will contain only Index Sorted Data) (Figure 5.95).
Figure 5.95 Default Plate Heat Map with GFP Arithmetic Mean Y Parameter
3. Insert a Color Dot Plot.
4. Change the axes on the color dot plot to the parameters of interest.
5. Create a Well Gate on all wells.
Notes:
o In addition to creating a gate on all wells a gate may be created on one, or any number of wells, to visualize the individually sorted cells.
o Since Influx Index Sorting Data Files also contain data for the unsorted events, we can use the Emphasize on Plots feature of gates to differentiate the sorted from non sorted events (Figure 5.96).
Figure 5.96 Plate heat map with Well Gate created on all wells. Standard gate is shown above while a gate with the Emphasize on Plots check box checked is shown below. Note that events highlighted in green fall within the well gate and in this case represent all events that were sorted into the plate.
6. Optional: Apply the Well Gate created in step 3 to the color dot plot to only view Index Sorted Events (Figure 5.97).
Figure 5.97 Color Dot Plot gated on Well Gate with events emphasized
It is possible to insert a plate heat map statistics grid in a text box to display statistics for each well in the plate heat map as done in Figure 5.98.
Figure 5.98 Plate Heat Map Statistics Grid displaying statistics for the Y parameter of the Plate Heat Map
Another useful feature for index sorting data is the Plate Heat Map Overlay function. Plate Heat Map Overlays allow a user to view multiple statistics and parameters on a single plate heat map by adding each statistic as a new overlay. For instance, in Figure 5.99 left, the upper slice of the well represents PE-CY7 arithmetic mean while the lower slice represents GFP arithmetic mean so single and double positive events can be more easily visualized.
Additionally, users may add a Radius Statistic, which changes the size of the well to reflect the selected parameter. For instance, in Figure 32 below (right), the heat/color for each well is dependent on the GFP arithmetic mean while the size of the well radius is dependent on the PE-Cy7 arithmetic mean.
Please see the Plate Heat Map Overlay section of the manual for full instructions on how to create a plate heat map overlays of different parameters and types as well as setting the radius parameter statistic.
Figure 5.99 Custom Plate Heat Maps with Overlay (left) and Radius Parameter (right). Standard Deviations of Color Levels was changed to 1.