Phospho flow cytometry methods for the analysis of kinase signaling in cell lines and primary human blood samples.
Methods Mol Biol
; 699: 179-202, 2011.
Article
em En
| MEDLINE
| ID: mdl-21116984
ABSTRACT
Phospho-specific flow cytometry, or phospho flow, measures the phosphorylation state of intracellular proteins at the single cell level. Many phosphorylation events can be analyzed simultaneously in each cell, along with cell surface markers, enabling complex biochemical signaling networks to be resolved in heterogeneous cell populations. The method has been applied to many diverse areas of biology, including the characterization of signaling pathways in normal immune responses to antigenic stimulation and microbial challenge, alteration of signaling networks that occur in cancer and autoimmune diseases, and high-throughput, high-content drug discovery. In this chapter, we provide detailed experimental protocols for performing phospho flow in cell lines, Ficoll-purified peripheral blood mononuclear cells, and whole blood. These protocols are applicable to both human and murine samples. We also provide methods for the validation of surface marker antibodies for use in phospho flow. Finally, we discuss data analysis methods, in particular, how to quantify changes in phosphorylation and how to visualize the large data sets that can result from experiments in primary cells.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfotransferases
/
Transdução de Sinais
/
Citometria de Fluxo
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Estados Unidos