Gating

Overview of Gating

Definition: Gating is the process of selecting specific cell populations for analysis based on their expression of cell surface markers, their size, and their granularity
Purpose:
- Identify Cell Populations: To identify and quantify different cell types in a sample
- Reduce Background Noise: To exclude debris, dead cells, and other unwanted events from the analysis
- Isolate Subsets: To isolate subsets of cells based on their expression of multiple markers
Key Concepts:
- Gates
- Regions
- Hierarchical Gating
- Boolean Gating

Definition: A boundary drawn on a flow cytometry plot that is used to select a specific population of cells
Types of Gates:
- Rectangle Gates: A rectangular region defined by two x-axis values and two y-axis values
- Ellipse Gates: An elliptical region defined by a center point, a major axis, a minor axis, and an angle of rotation
- Polygon Gates: A polygonal region defined by a series of vertices
- Freeform Gates: A region that is drawn manually using a freeform shape
Considerations:
- Gate Placement: The placement of the gate should be based on the expression of the cell markers and the distribution of the data
- Gate Size: The size of the gate should be large enough to include all of the cells in the population of interest, but small enough to exclude unwanted events
- Controls: Use appropriate controls to validate the gating strategy and to account for background noise

Definition: An area on a flow cytometry plot that is used to define a specific cell population
Difference Between Gates and Regions:
- Gates are used to select cells for further analysis, while regions are used to define cell populations
- Gates are typically applied to listmode data, while regions are typically used to analyze histograms
Types of Regions:
- Marker Regions: Regions that are defined based on the expression of a single marker
- Quadrant Regions: Regions that are defined by two perpendicular lines, dividing the plot into four quadrants
- Multi-Dimensional Regions: Regions that are defined based on the expression of multiple markers
Setting Quadrant Gates:
- Set the gates based on the FMO to determine what the cut off values will be.
- This may be done using an unstained control.
General notes on setting gates:
- When setting gates on an entire population of cells, review the samples to ensure that the gate is properly placed

Definition: A gating strategy in which cells are selected based on a series of sequential gates, with each gate defining a subset of the cells selected by the previous gate
Purpose:
- Identify Complex Cell Populations: To identify cell populations that are defined by the expression of multiple markers
- Reduce Background Noise: To exclude unwanted events from the analysis
Implementation:
1. Start with a broad gate to select all of the cells of interest
2. Apply subsequent gates to select subsets of cells based on their expression of additional markers
3. Continue gating until the desired cell population is isolated
Example:
- First, gate on lymphocytes based on forward and side scatter properties
- Then, gate on CD3+ T cells
- Then, gate on CD4+ helper T cells
- Then, gate on CD25+FoxP3+ regulatory T cells

Definition: A gating strategy in which cells are selected based on the combination of multiple gates using Boolean logic operators
Purpose:
- Define Complex Cell Populations: To define cell populations that are based on the expression of multiple markers
- Identify Overlapping Populations: To identify cells that express multiple markers simultaneously
Boolean Operators:
- AND: Selects cells that are within all of the specified gates
- OR: Selects cells that are within any of the specified gates
- NOT: Selects cells that are not within the specified gate
Example:
- Select cells that are CD3+ AND CD4+ AND CD8-
- Select cells that are CD19+ OR CD20+

Use Appropriate Controls:
- Use isotype controls, FMO controls, and single-stain controls to validate the gating strategy and to account for background noise
Base Gate Placement on Biology:
- The placement of the gates should be based on the known biology of the cells being studied
- Consult the literature and use appropriate cell markers to identify the cell populations of interest
Use a Consistent Gating Strategy:
- Use the same gating strategy for all of the samples in the experiment to ensure consistency
Document the Gating Strategy:
- Document the gating strategy in detail, including the markers used, the gate names, and the rationale for the gate placement
Apply the same strategy to all samples:
- Any changes to the strategy or gate placement must be well documented

Overlapping Populations:
- Possible Causes:
  - Poor resolution
  - Incorrect compensation
  - Inappropriate markers
- Troubleshooting Steps:
  - Optimize staining protocol
  - Adjust instrument settings
  - Verify compensation settings
  - Select more appropriate markers
Low Event Count:
- Possible Causes:
  - Incorrect gating
  - Sample loss
  - Rare cell population
- Troubleshooting Steps:
  - Review and adjust the gating strategy
  - Optimize sample preparation
  - Increase sample volume
Unexpected Results:
- Possible Causes:
  - Incorrect antibody selection
  - Improper gating
  - Instrument malfunction
- Troubleshooting Steps:
  - Verify antibody specificity
  - Review gating strategies
  - Inspect instrument for malfunctions

Gating: The process of selecting specific cell populations for analysis based on their expression of cell surface markers, their size, and their granularity
Gate: A boundary drawn on a flow cytometry plot that is used to select a specific population of cells
Region: An area on a flow cytometry plot that is used to define a specific cell population
Hierarchical Gating: A gating strategy in which cells are selected based on a series of sequential gates
Boolean Gating: A gating strategy in which cells are selected based on the combination of multiple gates using Boolean logic operators
Control Samples: Samples used to validate the gating strategy and to account for background noise