Data acquired using any guavaSoft™ module other than InCyte™, for example, ExpressPro, will not have an Analysis Method associated with it. However, when you open the file in InCyte™, a default Method will automatically be created.
1 From the analysis pane, choose File > Open from the menu bar. Select an FCS file
for analysis and click Open. You can also use the Open Group icon in the Data pane
or the Open Analysed Group icon in the Analysed Data pane. You can also drag and
drop an FCS file from a folder to the Data pane.
The data for the first sample well appears. For information on selecting the number and type of plots (dot plot, histogram, or contour plot) you wish to display, and changing the plot parameters, see “Plots” on page 4-2.
Click the + to the left of the data file name to display a list of all sample wells and tubes contained in the file. All wells with acquired data appear as open circles in the corresponding locations in the plate map. Data for any well can be displayed in the plots by selecting the sample from the file list or clicking on a well in the plate map.
2 When a data file is generated in a guava® software module other than InCyte™,
InCyte™ will automatically create a default Analysis Method and Analysed Group for the data file/set. The Method will contain a percent (%) metric, initially. If you wish to open an existing Method use the Open Method icon. Method files have the extension
.gsy. To rename the Method, double-click it and enter a new name related to this analysis.
3 If you opened an existing Method, you will need to drag it to the Analysed Group.
Notice the sample wells in the plate map become dark blue (if the Method was newly created) or various shades of blue (if a previously defined Method is used). You can place the cursor over any well in the plate map to view results for that sample.
Clicking on a sample in the Data pane will display the unanalyzed data for that sample in the plots, however the information in the plate map will be lost. Click on the
Analysed Group to redisplay the information in the plate map. Click on the individual Click New Method to create a new Method.
Analysis Method containing a metric, gate list, and region list.
Data and Method are automatically paired to create an Analysed Group. If you open a saved Method, you will need to drag it to the Analysed Group.
wells in the plate map to display the analyzed data for that well in the plots, or use the arrow keys to quickly scroll through the wells, displaying the data.
NOTE:
Any data file or Method file can be replaced by simply selecting it from the respective pane and dragging to the Analyzed Group. This allows you to quickly interchange data files and Methods.To save an AnalysedGroup, click on it in the Analysed Data pane to select it, then click the Save Analysed Group icon. Navigate to the location, enter a name, and click
Save. The AnalysedGroup will be saved as an FCS file and will contain the method
(regions, gates, metrics, and stats), as well as the data and the instrument settings.
4 You are now ready to start customizing your Analysis Method using regions and
gates. Refer to “Regions, Gates, and Statistics” on page 4-19.
Regions, Gates, and Statistics
Regions
InCyte™ offers six types of regions/markers—ellipses, rectangles, octagons, polygons, and quadrant markers for dot plots, and histogram markers for histograms. All of these regions are created using either the New Region icon in the plot tool bar (see “Plots” on page 4-2), except the quadrant marker, or the New Stat Marker icon in the tool bar, which allows you to select statistics for the region/marker. For information on creating
quadrants, refer to “Stat Markers” on page 4-26. A single Analysis Method can contain a maximum of 24 regions or stat markers, and you can have multiple stats per marker. InCyte™ regions are associated with the one (histogram) or two (dot plot) parameters in which they were created. If you change the x- or y-axis parameter on the plot, the region is no longer displayed. However, it is still contained within the Method.
1 Once an AnalysedGroup is created, either by pairing a data file with a Method or
dragging the AnalysedGroup to the legend, click the New region icon in the plot tool
bar and select a region from the pop-up menu.
You are prompted to enter a region name. The default is R1. When creating a polygon, this dialog appears after you create the region.
2 Enter a name for the region and click OK.
You may leave it R1, but a unique region name can be helpful in differentiating Analysis Methods.
The newly created region appears on the plot with the region name. Click and drag the name to move it. The region is listed in the Region List under the Method (see below) and in the Region List table (Region List icon in the main tool bar or Tools > Show Region List). To delete a region, see “Region List” on page 4-21.
3 Adjust the region to encompass the data. Or, for a polygon, create the region.
• To adjust an elliptic region, click anywhere on the edge
of the ellipse, except on a handle, and drag it to a new location. The ellipse has two handles. The open circle allows you to narrow/widen the ellipse. The solid circle allows you to lengthen, as well as rotate the ellipse around a point opposite the solid circle.
• To adjust a rectangle region, click anywhere on the
edge of the rectangle, except on a handle, and drag it to a new location. The rectangle has five handles. The open squares allow you to extend at the corresponding corner. The solid square allows you to rotate and resize.
• To adjust an octagonal region, click anywhere on the
edge of the octagon, except on a handle, and drag it to a new location. The octagon has eight handles. Click and drag the handles to adjust the shape of the octagon. To add or remove segments, right-click on a handle and select Insert Line Segment or Delete Line Segment.
• To create a polygon region, click to add the first
handle, then move the cursor to the next point and click. Continue creating up to 32 segments until the shape is complete. Finish by clicking again at the first handle to close it. To move it, click anywhere on the edge, except on a handle, and drag it to a new location. To adjust the shape, click and drag the handles. To resize or rotate, press the Shift key and click and drag a handle.
• To adjust a histogram region, click either of the two
handles. The histogram marker can be moved vertically as well as horizontally.