FCS Express: Index Sorting

Working with Index Sorting Data

Index sorting allows researchers to review the complete phenotype of every single event sorted into a plate and associate that event with all sorted and non sorted events. FCS Express allows for analysis and reporting of index sorting data using Heat Maps along with the standard plot offerings.

FCS Express currently supports index sorting data files from the following instruments (please click links for full support documentation):

See below for individual examples from instrument specific data files.

Combining Heat Maps and Color Dot Plots to Analyze Index Sort Data

For index sort data, Heat maps will display the number of events (usually one) and parameter based phenotypic data for all sorted events. Wells may be gated on individually, or as groups, with the well gates backgated onto other plots to view the individually sorted cells in context of the entire acquired/sorted samples. All heat map functionality is available for use with index sorting data. FCS Express offers a number of features, including Emphasized Well Gates, to properly analyze Index Sort Data.

Standard Well Gate versus Emphasized Well Gate on Influx Index Sorting Data

 

Customizing Heat Maps for Index Sort Data

Heat Map Overlays allow a user to view multiple statistics and parameters on a single heat map by adding each statistics as a new overlay. Additionally, users may add a radius statistic, which changes the size of the well to reflect the selected parameter.

Heat Map with Overlay
Heat Map with Radius Statistic
Lower Well Statistic: GFP Arithmetic Mean

Upper Well Statistic: PE-CY7 Arithmetic Mean

Heat Statistic: GFP Arithmetic Mean

Radius Statistic: PE-CY7 Arithmetic Mean

Instrumentation Examples

Influx Index Sorting

    Data File Information:

    Influx Index Sorting Data Files contain fluorescence information on both the sorted and unsorted events. A default heat map can be created to display statistics related to a single parameter or a custom heat map can be created to display multiple parameters related to the index sort data. Well gates can be created and applied to view index sorting data on a color dot plot. Individual well gates can be created to isolated a single sorted event. In addition, a well gate may be created on a group of wells to isolate multiple sorted events.
Heat Map with Radius Parameter
Heat Statistic: GFP Arithmetic Mean

Radius Statistic: PE-CY7 Arithmetic Mean

Index Sorted and Non Sorted Events
All Index Sorted Events
Single Index Sorted Event

 

Diva Index Sorting

    Data File Information:

    Diva Index Sorting Data Files contain fluorescence information only on the indexed sort events. A "master" .fcs file contains information on all of the events collected during a sort. These two files may be used individually or in conjunction via plot overlays to help visualize index sorting data acquired with Diva. A default heat map can be created to display statistics related to a single parameter or a custom heat map can be created to display multiple parameters related to the index sort data. Well gates can be created and applied to view index sorting data on a color dot plot. Individual well gates can be created to isolated a single sorted event. In addition, a well gate may be created on a group of wells to isolate multiple sorted events.
Heat Map with Radius Parameter
Heat Statistic: FITC Arithmetic Mean

Radius Statistic: PE Arithmetic Mean

Index Sorted Data File
"Master" Data File
Index Sorted and "Master" File Overlay

Summit Software Sorting Data

    Data File Information:

    Summit Software Sorting Data Files contain fluorescence information on both the sorted and unsorted events. A default heat map can be created to display statistics related to a single parameter or a custom heat map can be created to display multiple parameters related to the index sort data. Well gates can be created and applied to view index sorting data on a color dot plot. Individual well gates can be created to isolated a single sorted event. In addition, a well gate may be created on a group of wells to isolate multiple sorted events.
Heat Map with Overlay
Lower Well Statistic: FL5 Arithmetic Mean

Upper Well Statistic: FL13 Arithmetic Mean

Index Sorted and Non Sorted Events
All Index Sorted Events
Single Index Sorted Event

 

Chorus Index Sorting

    Data File Information:

    Chorus Index Sorting Data Files only allow for analysis on indexed sorted events. A default heat map can be created to display statistics related to a single parameter or a custom heat map can be created to display multiple parameters related to the index sort data. Well gates can be created and applied to view index sorting data on a color dot plot. Individual well gates can be created to isolated a single sorted event. In addition, a well gate may be created on a group of wells to isolate multiple sorted events.
Heat Map with Overlay
Lower Well Statistic: PerCP Arithmetic Mean

Upper Well Statistic: FITC Arithmetic Mean

Index Sorted Events
Index Sorted Events with Well Gates Applied