Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications

被引:232
作者
Krutzik, PO
Irish, JM
Nolan, GP
Perez, OD
机构
[1] Stanford Univ, Sch Med, Dept Microbiol & Immunol, Baxter Lab Genet Pharmacol, Stanford, CA 94305 USA
[2] Stanford Univ, Sch Med, Dept Mol Pharmacol, Stanford, CA 94305 USA
关键词
intracellular; phosphorylation; multiparameter; phospho-specific antibodies; activation; drug screening; disease characterization;
D O I
10.1016/j.clim.2003.11.009
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
Analysis of protein phosphorylation with flow cytometric techniques has emerged as a powerful tool in the field of immunological signaling, allowing cellular subsets in complex populations to be analyzed accurately and rapidly. In this review, we examine the development of phospho-epitope, or phospho-specific, flow cytometry and the premises upon which the technique is based. Phospho-specific flow cytometry is compared to traditional biochemical methods, and its advantages, such as single cell analysis, multiparameter data acquisition, rapid protocols, and the ability to analyze rare cell subsets, are detailed. We also discuss the many technical considerations that must be addressed when developing new antibodies or analyzing new epitopes including antigen accessibility, stability of the phosphoepitope, fluorophore selection, surface phenotype integrity, and antibody suitability for staining epitopes inside fixed and permeabilized cells. The methods that have been used to date are described in light of these technical considerations. The importance of developing bioinformatic platforms in parallel with these techniques is emphasized due to the large, multiparameter datasets that are rapidly accumulated and which require more efficient data viewing and complex clustering methods than currently available for flow cytometric data. Finally, we discuss the potential clinical applications of phospho-specific flow cytometry in analyzing immune cell development and antigen-specific immune responses, as well as pharmacodynamic profiling of disease states or drug efficacy and specificity against particular signaling proteins. (C) 2004 Elsevier Inc. All rights reserved.
引用
收藏
页码:206 / 221
页数:16
相关论文
共 82 条
[41]   Quantitation, selection, and functional characterization of Epstein-Barr virus-specific and alloreactive T cells detected by intracellular interferon-γ production and growth of cytotoxic precursors [J].
Koehne, G ;
Smith, KM ;
Ferguson, TL ;
Williams, RY ;
Heller, G ;
Pamer, EG ;
Dupont, B ;
O'Reilly, RJ .
BLOOD, 2002, 99 (05) :1730-1740
[42]   Evaluation of caspase activity in apoptotic cells [J].
Köhler, C ;
Orrenius, S ;
Zhivotovsky, B .
JOURNAL OF IMMUNOLOGICAL METHODS, 2002, 265 (1-2) :97-110
[43]   Intracellular phospho-protein staining techniques for flow cytometry: Monitoring single cell signaling events [J].
Krutzik, PO ;
Nolan, GP .
CYTOMETRY PART A, 2003, 55A (02) :61-70
[44]   FLOW CYTOMETRIC MULTIPARAMETER ANALYSIS OF PROLIFERATING CELL NUCLEAR ANTIGEN CYCLIN AND KI-67 ANTIGEN - A NEW VIEW OF THE CELL-CYCLE [J].
LANDBERG, G ;
TAN, EM ;
ROOS, G .
EXPERIMENTAL CELL RESEARCH, 1990, 187 (01) :111-118
[45]   Studies of human antiviral CD8+lymphocytes using class I peptide tetramers [J].
Lechner, F ;
Cuero, ALV ;
Kantzanou, M ;
Klenerman, P .
REVIEWS IN MEDICAL VIROLOGY, 2001, 11 (01) :11-22
[46]  
Lechner F, 2000, EUR J IMMUNOL, V30, P2479, DOI 10.1002/1521-4141(200009)30:9<2479::AID-IMMU2479>3.0.CO
[47]  
2-B
[48]   Multiparametric flow cytometric analysis of biochemical and functional events associated with apoptosis and oncosis using the 7-aminoactinomycin D assay [J].
Lecoeur, H ;
de Oliveira-Pinto, LM ;
Gougeon, ML .
JOURNAL OF IMMUNOLOGICAL METHODS, 2002, 265 (1-2) :81-96
[49]   A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes [J].
Lecoeur, H ;
Ledru, E ;
Gougeon, ML .
JOURNAL OF IMMUNOLOGICAL METHODS, 1998, 217 (1-2) :11-26
[50]   Multiparametric flow cytometric analysis in a breast cancer cell line (MCF-7) [J].
Lee, GSR ;
Ryu, KS ;
Rha, JG ;
Kim, SP ;
Namkoong, SE ;
Han, KT .
JOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH, 2002, 28 (03) :141-148