Backgating in Flow Cytometry

Author: Mike Blundell | Reviewer: Chloe Fenton

What Is Backgating?

Backgating is a useful method of identification of cells to confirm a staining pattern or gating method. It allows you to analyze cells identified in a gate on dot plots with different parameters. This can be useful if you are unsure of your gates, the expression levels, nonspecific binding, or whether you have identified your cells of interest or the presence of dead cells and need additional information to identify your cells.

Use Example: Backgating to Confirm Leukocyte Subsets

An example of backgating in action can be seen in Figure 18.

Fig. 18. Backgating to identify leukocyte subsets. A. Red cell lysed whole blood. B. Stained for CD3 (MCA463A647) and CD14 (MCA1568P750). C. Cells in the green, blue, and red gates were backgated onto FSC vs. SSC to confirm leukocyte populations.

Practical Guidance

Gating does not need to be a daunting process and by following just a few simple steps you can quickly begin to analyze specific cell populations. As you increase the number of stains and fluorophores you will be able to identify more specific cell populations. However, make sure you perform the right controls and have an adequate sample size because as you increase the fluorescence you increase the background and nonspecific binding, making the data harder to analyze.

How To Use Backgating

1. Select a population of interest.
   Start with cells already identified in a gate on a dot plot (a “daughter” population).
2. Choose a second plot to validate identity.
   Open a plot using different parameters (for example, FSC vs SSC) to provide additional context.
3. Project the gated cells onto the new plot.
   Backgate the selected daughter population onto the second plot to see where those events fall.
4. Confirm expected location and separation.
   Check whether the backgated cells cluster where expected for that population; use this to confirm staining patterns or gating methods and to identify issues such as debris or mixed populations.
5. Refine gates and review controls if needed.
   If backgated events appear scattered, overlapping, or inconsistent with expected regions, revise the gating strategy and review controls (and, where relevant, compensation or unmixing) before proceeding.