Gating in flow cytometry is the process of identifying populations of cells for analysis through a series of selecting populations of interest in "gates". Manual gating is based on the knowledge of the cellular characteristics (i.e. forward and side scatter profiles and for the presence or absence of a marker). In addition to identifying your cells based on prior knowledge of their markers, manual gating is also often employed to cleanup your data (e.g. gating on live cells) before performing high dimensional analysis.
Gating Nomenclature in OMIQ
Filter: Another term for gate
Gating Tree: Refers to the gating hierarchy (displayed on the left of the plot in the gating task)
Node: Refers to a filter (gate) in the gating tree
Branch: Refers to the parent node and all its associated children node in the gating tree
Symbols in the Gating Tree:
1. This is a filter (gate) in your gating hierarchy. This is referred to as a Node in OMIQ gating tree.
2. Identifies the tree node that is the current active filter for the data displayed in the plot.
3. The gate for this tree node is currently visible in the plot.
4. This brings up the Gating Tree options menu.
1. Add a Gating Task
Click Add new child task and select Gating from the task selector.
Note that your Workflow might look slightly different, if you are using Compact View.
2. Build your Gating Tree
2.1 Draw a Gate
Create a gate in your plot by selecting a Gate Option. You can change the x and y axis features by clicking on the Drop-down Arrow.
Gate Options in OMIQ
1. Rectangle Gate: Draws a rectangle gate on the current plot.
Rectangle Gates can be converted to a polygon by right clicking the rectangle gate and selecting Convert to polygon.
2. Polygon Gate: Allows you to draw a free-form polygon on the current plot.
Click on the first point of your polygon to finalize your gate.
3. Ellipse Gate: Draws an ellipse gate on your plot.
4. Quadrant Gate: Easily divide your plot into four quadrants.
The quadrants created using the Quadrant Gate are not independently adjustable from each other. If you need to have an adjustable quadrant, use Skewed Quadrant Gate. To convert a quadrant gate to a skewed quadrant gate, right click on the center point of the quadrants and select Convert to skewed quadrant.
5. Skewed Quadrant Gate: Allows you to create a quadrant gate that has quadrants you can adjust independently from each other.
If you want to straighten the arms of the skewed quadrant gate, right click on any of the points in the skewed quadrant gate and select Straighten arms.
6. Range Gate: Allows you to select a region within your plot. Normally used in Histograms.
7. Split Gate: Splits your plot into positive and negative areas. Normally used in Histograms.
2.2 Build a Gating Tree Hierarchy
You can start to create a gating tree hierarchy by drilling down on existing gates. Double-click on an existing gate to drill down on the chosen population.
In the image above we have identified our cells by using the polygon gate. We have double-clicked on the gate to activate the Cells gate and focus on the gated population.
You can also choose to activate a particular node in the gating tree by right clicking on the node of interest and selecting Activate.
2.3 Applying Gates to a Single File or Metadata Group
OMIQ gates are applied to all files automatically. Any change in a gate will be applied to all files. To fine tune your gating, you can use Per-File Positioning or Metadata Positioning.
- Per-File Positioning allows you to adjust gates independently on each file.
- Metadata Positioning allows you to adjust gates per metadata grouping.
Right clicking on any gate will bring up choices for either Enable per-file positioning or Enable metadata positioning. You can also access these in the Gating Tree Options.
You can create new gates on a per-file basis by selecting the Per-file mode on for new gates in the Plot Settings
2.4 Making Boolean Filters
Right click on a Node in the Gating Tree and select Boolean Filters. Available Boolean filters are NOT, OR, AND, or Combo filters.
2.5 Copying Gates
To copy or cut a gate, right click on a Node in your gating tree and select Copy/Cut.
To paste a copied or cut gate, right click on a Node in your gating tree and select Paste, which pastes the gate as a child.
OMIQ allows you to paste nodes as either unlinked or linked (Paste Linked option). Nodes pasted as linked applies changes in one node to all linked nodes.
To paste a copied or cut gate to multiple nodes, select the Gating Tree options (three dots) and select Bulk Paste.
2.6 Renaming Gates
To rename a gate, right click on a Node and select Rename.
You can also rename a rectangle, polygon, ellipse, or range gate on your plot by right clicking on the gate and selecting Rename.
2.7 Deleting Gates
To delete a gate, right click on a Node in the Gating Tree and select Delete allowing you to delete a node, a branch, or children nodes.
Deleting a node with children nodes does not delete any children nodes. The children nodes become children of the grandparent node. To delete a node and associated children nodes, you need to select Delete Branch.
You can also delete a gate by right clicking on the gate in the plot and selecting Delete gate.