Protocol

Single Cell Protein Analysis

Volume 1346 of the series Methods in Molecular Biology pp 99-113

Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry

  • Nalin LeelatianAffiliated withCancer Biology, Vanderbilt University School of Medicine
  • , Kirsten E. DigginsAffiliated withCancer Biology, Vanderbilt University School of Medicine
  • , Jonathan M. IrishAffiliated withCancer Biology, Vanderbilt University School of MedicinePathology, Microbiology and Immunology, Vanderbilt University School of Medicine Email author 

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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/​).

Key words

Single cell biology Mass cytometry (CyTOF) Human Immunophenotyping Signaling network profile Phospho-specific flow cytometry (phospho-flow)