Flow cytometry is a technology used to rapidly analyze cells or particles in a buffered salt-based solution. Flow cytometry uses one, or multiple, lasers to determine the visible light scatter given off by each cell. This light scatter is measured in two specific directions, forward and sideways, which provide information on the relative size of the cell and the internal complexity or granularity of the cell. Today, flow cytometers with multiple lasers are most common, and some instruments are even equipped for a specific purpose (e.g., coupled to a mass spectrometer). The latest generations of clinical flow cytometers include increasingly advanced scanning abilities, and can analyze cells at rates above 20,000 cells/second.

Prior to flow cytometry, samples must be stained or probed to allow for detection, which can be done in a number of ways. For example, cells can be transfected with recombinant fluorescent proteins like green fluorescent protein (GFP), stained with fluorescent dyes like propidium iodide (PI), or conjugated with fluorescent antibodies like CD3 or FITC. The availability and diversity of fluorochromes has also increased over the years, which in turn increased the number and type of analysis parameters available to detect. New recombinant fluorescent proteins have evolved from GFP into many different variants, like mCherry, mBanana, mOrange, and mNeptune.

The use of tandem dyes and polymer dyes has also helped increase the capacity of fluorochromes capable of conjugating to monoclonal antibodies. Data analysis techniques have also advanced from the traditional two parameter histogram (dot plot) gating methods to complex cluster data analysis, able to provide high-dimensional data.

Often, flow cytometry related issues are not apparent until after the data after the sample has already been tested. Some common issues, troubleshooting tips, and possible solutions are listed below.