Gating is the process of selecting the target cells to be sorted by applying the correct charge to the droplets in order to send them to the collection tubes after passing by the deflection plates. Gates can be set depending on FSC/SSC values or fluorescence intensity.

The most common first gating strategy is to use FSC and SSC to find viable, single cell events and eliminate any debris, dead cells, and clumps or doublets. Normally, dead cells often have lower FSC and higher SSC than live cells.

Once target cells are identified using FSC and SSC, they can be further selected according to their fluorescence. If there is only one fluorescent marker, a univariate histogram is displayed, while if two different markers are used, graphs are normally plotted as a bivariate histogram (Figure 1). This divides the plot into four quadrants for the four possible combinations: double positive, single positive for each fluorescent marker, and negative for both.

Figure 1. Bivariate histogram.