Backgating to Confirm Gating Strategies

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, non-specific binding, whether you have identified your cells of interest or the presence of dead cells and need additional information to identify your cells. This can be seen in Figure 18.

Fig. 18. Backgating to identify leukocyte subsets. A. Red cell lyzed 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.

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 non-specific binding, making the data harder to analyze.