High‐dimensional cytometry represents an exciting new era of immunology research, enabling the discovery of new cells and prediction of patient responses to therapy. A plethora of analysis and visualization tools and programs are now available for both new and experienced users; however, the transition from low‐ to high‐dimensional cytometry requires a change in the way users think about experimental design and data analysis. Data from high‐dimensional cytometry experiments are often underutilized, because of both the size of the data and the number of possible combinations of markers, as well as to a lack of understanding of the processes required to generate meaningful data. In this article, we explain the concepts behind designing high‐dimensional cytometry experiments and provide considerations for new and experienced users to design and carry out high‐dimensional experiments to maximize quality data collection.
Microglia, the resident innate immune cells of the central nervous system (CNS), play an important role in brain development and homoeostasis, as well as in neuroinflammatory, neurodegenerative and psychiatric diseases. Studies in animal models have been used to determine the origin and development of microglia, and how these cells alter their transcriptional and phenotypic signatures during CNS pathology. However, little is known about their human counterparts. Recent studies in human brain samples have harnessed the power of multiplexed single‐cell technologies such as single‐cell RNA sequencing (scRNA‐seq) and mass cytometry (cytometry by time‐of‐flight [CyTOF]) to provide a comprehensive molecular view of human microglia in healthy and diseased brains. CyTOF is a powerful tool to study high‐dimensional protein expression of human microglia (huMG) at the single‐cell level. This technology widens the possibilities of high‐throughput quantification (of over 60 targeted molecules) at a single‐cell resolution. CyTOF can be combined with scRNA‐seq for comprehensive analysis, as it allows single‐cell analysis of post‐translational modifications of proteins, which provides insights into cell signalling dynamics in targeted cells. In addition, imaging mass cytometry (IMC) has recently become commercially available, and will be useful for analysing multiple cell types in human brain sections. IMC leverages mass spectrometry to acquire spatial data of cell–cell interactions on tissue sections, using (theoretically) over 40 markers at the same time. In this review, we summarise recent studies of huMG using CyTOF and IMC analyses. The uses and limitations as well as future directions of these technologies are discussed.
Myeloid cells are suggested as an important player in Alzheimer´s disease (AD). However, its continuum of phenotypic and functional changes across different body compartments and their use as a biomarker in AD remains elusive. Here, we perform multiple state-of-the-art analyses to phenotypically and metabolically characterize immune cells between peripheral blood (n = 117), cerebrospinal fluid (CSF, n = 117), choroid plexus (CP, n = 13) and brain parenchyma (n = 13). We find that CSF cells increase expression of markers involved in inflammation, phagocytosis, and metabolism. Changes in phenotype of myeloid cells from AD patients are more pronounced in CP and brain parenchyma and upon in vitro stimulation, suggesting that AD-myeloid cells are more vulnerable to environmental changes. Our findings underscore the importance of myeloid cells in AD and the detailed characterization across body compartments may serve as a resource for future studies focusing on the assessment of these cells as biomarkers in AD.
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