Immune Cell Phenotyping Using Flow Cytometry

Abstract Fluorescent immunophenotyping uses fluorescently‐conjugated antibodies to identify, characterize and quantify distinct subpopulations of cells within heterogeneous single‐cell populations, either in the context of tissue (using fluorescence and imaging microscopy) or in a single‐cell suspension (using multiparameter imaging microscopy, imaging cytometry, and/or flow cytometry). Flow cytometry is an optical, laser‐based technology which analyzes the physical and fluorescent properties of cells in suspension in real‐time as they flow through the instrument. This approach has a number of advantages over other techniques that can be used for characterizing cell populations in single‐cell suspensions, in that it can nonsubjectively interrogate up to millions of cells and acquire data on the presence of different cell subpopulations and phenotypical changes within these populations in seconds. This unit describes basic procedures for the direct and indirect immunofluorescent staining of surface and intracellular proteins that are expressed by lymphoid cells which have been isolated from tissues or blood. Protocols for the resolution of dead cells and for the fixation of cells are also included. This unit also provides essential information relating to the selection and titration of antibodies, fluorochrome choice, spectral overlap and compensation, the use of controls, and the standardization of data acquisition and analysis. It also highlights new technologies and platforms that can be used to interrogate the presence of cell subpopulations and their phenotype to an even greater depth. © 2015 by John Wiley & Sons, Inc.