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Flow cytometric analysis of intracellular phosphoproteins in human monocytes
Author(s) -
Coppin Emilie,
Malergue Fabrice,
Thibult MarieLaure,
Scifo Caroline,
Favre Cédric,
Nunès Jacques A.
Publication year - 2017
Publication title -
cytometry part b: clinical cytometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 61
eISSN - 1552-4957
pISSN - 1552-4949
DOI - 10.1002/cyto.b.21207
Subject(s) - phosphorylation , cd14 , flow cytometry , microbiology and biotechnology , mapk/erk pathway , intracellular , stimulation , biology , protein kinase b , signal transduction , chemistry , endocrinology
Background Using antibodies against intracellular phosphoproteins, flow cytometry can be used to monitor simultaneously multiple signaling pathways. Here, we tested a recently released procedure to analyze phosphorylation events in human monocytes upon different types of stimulation. Methods Whole blood was treated by lipopolysaccharide (LPS) or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), then cells were labeled by antibodies recognizing cell surface and cytosolic proteins. Human monocytes were identified by a CD14 – CD45 staining and three phosphorylated proteins such as AKT, ERK‐1/2, and STAT5, were simultaneously detected by multicolor phosphoflow analysis. Results By this rapid method, we are able to detect directly from a blood sample several signaling events in human monocytes where LPS stimulation induces preferentially ERK‐1/2 phosphorylation where as GM‐CSF stimulation induces STAT5 phosphorylation. Conclusions This procedure provides a simultaneous measurement of multiple activated signaling molecules using a simplified and rapid protocol. © 2015 International Clinical Cytometry Society