title: Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry.
abstract: Single cell mass cytometry is revolutionizing our ability to quantitatively characterize cellular biomarkers and signaling networks. Mass cytometry experiments routinely measure 25-35 features of each cell in primary human tissue samples. The relative ease with which a novice user can generate a large amount of high quality data and the novelty of the approach have created a need for example protocols, analysis strategies, and datasets. In this chapter, we present detailed protocols for two mass cytometry experiments designed as training tools. The first protocol describes detection of 26 features on the surface of human peripheral blood mononuclear cells. In the second protocol, a mass cytometry signaling network profile measures 25 node states comprised of five key signaling effectors (AKT, ERK1/2, STAT1, STAT5, and p38) quantified under five conditions (Basal, FLT3L, SCF, IL-3, and IFNγ). This chapter compares manual and unsupervised data analysis approaches, including bivariate plots, heatmaps, histogram overlays, SPADE, and viSNE. Data files in this chapter have been shared online using Cytobank ( http://www.cytobank.org/irishlab/ ).
authors.affiliation: Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors.forename: Nalin
authors.initials: N
authors.lastname: Leelatian
authors.affiliation: Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors.forename: Kirsten E
authors.initials: KE
authors.lastname: Diggins
authors.affiliation: Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN, USA. jonathan.irish@vanderbilt.edu.|Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, 740B Preston Building, 2220 Pierce Avenue, Nashville, TN, 37232-6840, USA. jonathan.irish@vanderbilt.edu.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Irish
mesh_terms: D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism*; D010641:Phenotype; D010766:Phosphorylation; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods*
keywords: Human; Immunophenotyping; Mass cytometry (CyTOF); Phospho-specific flow cytometry (phospho-flow); Signaling network profile; Single cell biology