A Practical Guide to Flow Cytometry

Flow cytometry is a pivotal immunological technique renowned for its versatility in various biological applications, including immunophenotyping, intracellular cytokine analysis, proliferation assessment, apoptosis quantification, cell cycle investigation, signal transduction studies, and cell sorting. This review comprehensively examines the methodological framework essential for designing robust flow cytometry experiments. Key aspects covered include the formulation of appropriate buffers to maintain cell viability and ensure optimal antigen detection, meticulous selection and preparation of controls to validate experimental findings, strategic implementation of compensation techniques to mitigate spectral overlap in multicolor assays, systematic panel design for simultaneous detection of multiple biomarkers, and the judicious application of fluorescent stains for specific cellular components. By addressing these critical methodological elements, researchers can enhance the reliability and reproducibility of flow cytometric analyses, thereby advancing our understanding of complex biological processes. This review delineates the various reagents essential for the successful execution of flow cytometry experiments, including staining buffers, fluorochrome-conjugated antibodies, viability dyes, and fixation/permeabilization solutions. The review also discusses the computational aspects of flow cytometry, with particular emphasis on fluorescence compensation and the correction of spectral overlap between fluorochromes. Lastly, the review discusses the implementation of appropriate controls—such as fluorescence minus one (FMO), isotype, unstained, and biological controls—to ensure data accuracy, validate staining specificity, and identify potential artifacts.