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Flow cytometry is a powerful technique allowing multiparameter detection and quantification of single cells or particles including cell size, granularity, cell components (DNA, mRNA), surface receptors, intracellular proteins, and signaling events. The flow cytometer operates via three main systems: the fluidics, optics, and electronics, which work together to analyze the physical and chemical properties of your sample. The first system, the fluidics, transports your sample in a single stream through the instrument, from the sample tube, pass the lasers, and is either sorted for further experiments or discarded into the waste vessel. The second system, the optical system, is composed of a series of lasers; for excitation of your sample and attached fluorescence antibodies as it passes, a series of lenses; and a detector system. The third system is the electronic component, which enables the photocurrent from the detector system to be changed into electronic pulses to be processed by a computer and analyzed by flow cytometry software. Flow cytometry is thus a powerful technique, which is commonly used to determine the expression of cell surface markers and intracellular molecules to define cells into different populations by fluorescently labeled antibodies.The staining procedure outlined below creates a single-cell suspension for staining with a panel of flow cytometry antibodies, which target different surface markers, to identify an array of cell types. After staining the sample is loaded into the flow cytometer, where the fluorescently labeled cells are excited as they pass by the laser emitting light at various wavelengths which are detected by the flow cytometer. Each fluorescent antibody has its own excitation and emission spectrum allowing the use of multiple antibodies. However, the emission spectrums of different fluorochromes can overlap each other, called spectral overlap. Thus, it is important to have good compensation controls to eliminate any antibody spillover.The staining methods from this technique can be used for different cell types by changing the surface marker targeted by the flow antibody. It is also important to use knowledge of the density of surface molecule for detection and brightness of fluorochrome to guide antibody selection and also to titrate all antibodies prior to use.This chapter’s protocol has been designed specifically for detection of human CD8(+) T cells defining the activation status of the cells by surface marker phenotyping.