The essential basics of flow cytometry
For a better understanding we first want to illustrate the principles of flow cytometry (FCM). Flow cytometry has evolved over the last few decades and nowadays is a well-established technique that determines events based on their physical and/or chemical characteristics. Samples containing single events (classically cells) are injected into a flow cytometer and pass one or more laser beams in a fluid stream. The resulting light signals are then detected, quantified and displayed in a software after appropriate processing. Typically, depending on the type of flow cytometer used, between 10,000 and 40,000 events per second can be analyzed.
Classically, flow cytometry is used to measure cells and analyze different parameters such as surface protein expression, cell cycle or viability, but within recent years instruments and methods have been developed which also allow for the detection of much smaller particles such as extracellular vesicles (EVs) or viruses [reviewed in [1, 2]]. Flow cytometry was initially developed in the 1960s and especially after fluorochrome-conjugated antibodies have become available, it has been used for numerous applications for basic biological and especially immunological and hematological research. Three decades later, it reached clinical breakthrough with HIV and leukemia/lymphoma diagnostics becoming mainstream. Today, flow cytometry has become indispensable in research and clinical diagnostics and it is one of the most impressive and versatile techniques for characterizing single cells in a high-throughput, multi-parametric manner [reviewed in [3]].
For the sake of simplicity and for better understanding, we here first describe the basic principle of the technique of flow cytometry on cells [4]. As we will see later, the preliminary preparative and analytical methods suitable for analyzing extracellular vesicles are fundamentally different from that used for cells. The light signals generated by cells passing lasers lead to light scattering around the cell and also to the excitation of fluorescent dyes (fluorochromes), which then emit a corresponding light signal. As already mentioned, flow cytometric analyses of cells can be performed to collect a variety of different information, the most prevailing information being if a surface protein is present on the cellular surface or not, by using a fluorochrome-conjugated antibody specifically raised against that surface protein. Furthermore, cells can be also classified based on differences of how they scatter light. Blood cells, for example, can roughly be divided into three basic groups (lymphocytes, monocytes, and granulocytes) merely based on how they scatter light, since these cell types have different sizes and a different granularity (Fig. 1A).
