This signal is monitored and processed by the cytometer’s electronics, and is the origin of the “A”, “H”, and “W” pulse parameters we know and love ( Figure 1). Pulses occur as a cell pass through the laser beam spots, and this passage generates signal from the detectors. You may recall that an “event” is the fundamental unit of measurement in flow cytometry, and is defined by what we call a pulse. Given how good cytometers are at measuring single cells, how would a situation arise in which the instrument would not be able to do this? The answer to this question has a whole lot to do with cytometer electronics, and how they classify cells and other particles as events. In other words, the particles were so close together when they passed through this laser spot, that the instrument was incapable of distinguishing them as individual events or particles. The cytometer classified these particles as a single event because they passed through the interrogation point very close to one another.

Most straightforwardly, a doublet is a single event that actually consists of 2 independent particles. The first aspect to understanding how a doublet exclusion gate works is to define what a doublet is. You are probably familiar with the term, “doublet discrimination” or “doublet exclusion”, and have likely included this flow cytometry measurement into at least some (if not all) of your gating strategies.Įven though you may utilize this important gating strategy, you may not have had the chance to delve deeper to explore exactly what doublets are and why it’s critical to exclude them.