Staining of Phosphorylated Signalling Markers Protocol for Mass Cytometry

Diana Shinko; Thomas M Ashhurst; Helen M McGuire; Kellie A Charles

doi:10.1007/978-1-4939-9454-0_10

Staining of Phosphorylated Signalling Markers Protocol for Mass Cytometry

Methods Mol Biol. 2019:1989:139-146. doi: 10.1007/978-1-4939-9454-0_10.

Authors

Diana Shinko¹, Thomas M Ashhurst^{2

3

4

5}, Helen M McGuire^{6

7

8}, Kellie A Charles⁹

Affiliations

¹ Discipline of Pharmacology, School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia.
² Sydney Cytometry Facility, The University of Sydney and Centenary Institute, Camperdown, NSW, Australia.
³ Viral Immunopathology Laboratory, Discipline of Pathology, Faculty of Medicine and Health, School of Medical Sciences, Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia.
⁴ Ramaciotti Facility for Human Systems Biology (RFHSB), The University of Sydney and Centenary Institute, Sydney, NSW, Australia.
⁵ Marie Bashir Institute for Emerging Infectious Disease (MBI), The University of Sydney, Sydney, NSW, Australia.
⁶ Ramaciotti Facility for Human Systems Biology, The University of Sydney, Sydney, NSW, Australia.
⁷ Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia.
⁸ Discipline of Pathology, The University of Sydney, Camperdown, NSW, Australia.
⁹ Discipline of Pharmacology, School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia. kellie.charles@sydney.edu.au.

PMID: 31077104
DOI: 10.1007/978-1-4939-9454-0_10

Abstract

Mass cytometry is a multi-parametric technique that offers insight into functional and biological systems at a single cell level (Tanner et al., Cancer Immunol Immunother 62:955-965, 2013). One of the major advantages of mass cytometry is the ability to measure multiple intracellular markers, including phosphorylated proteins that are part of major signaling pathways, such as NF-κB, JAK/STAT, and ERK/MAPK. Here we describe an optimized mass cytometry protocol for staining human clinical blood samples with panels that include phosphorylated antibodies.

Keywords: Inflammatory pathways; Mass cytometry; Phosphorylation; Signaling pathway; Signaling proteins.

MeSH terms

Antibodies, Monoclonal / chemistry
Antibodies, Monoclonal / immunology
Biomarkers / analysis*
Flow Cytometry / methods*
Humans
Mass Spectrometry / methods*
Phosphoproteins / analysis*
Phosphoproteins / immunology
Phosphorylation
Single-Cell Analysis / methods*
Staining and Labeling / methods*

Substances

Antibodies, Monoclonal
Biomarkers
Phosphoproteins