Flow cytometric analysis of intracellular phosphoproteins in human monocytes.
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.



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.



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.



This procedure provides a simultaneous measurement of multiple activated signaling molecules using a simplified and rapid protocol. © 2015 International Clinical Cytometry Society.
Inserm, U1068, Centre de Recherche en Cancérologie de Marseille, Marseille, France.|Institut Paoli-Calmettes, Marseille, France.|CNRS, UMR7258, Centre de Recherche en Cancérologie de Marseille, Marseille, France.|Aix-Marseille Université, UM105, Marseille, France.
Emilie
E
Coppin
Beckman Coulter Immunotech, Life Sciences Global Assay and Applications Development, Marseille, France.
Fabrice
F
Malergue
Inserm, U1068, Centre de Recherche en Cancérologie de Marseille, Marseille, France.|Institut Paoli-Calmettes, Marseille, France.|CNRS, UMR7258, Centre de Recherche en Cancérologie de Marseille, Marseille, France.|Aix-Marseille Université, UM105, Marseille, France.
Marie-Laure
ML
Thibult
Beckman Coulter Immunotech, Life Sciences Global Assay and Applications Development, Marseille, France.
Caroline
C
Scifo
Beckman Coulter Immunotech, Life Sciences Global Assay and Applications Development, Marseille, France.
Cédric
C
Favre
Inserm, U1068, Centre de Recherche en Cancérologie de Marseille, Marseille, France.|Institut Paoli-Calmettes, Marseille, France.|CNRS, UMR7258, Centre de Recherche en Cancérologie de Marseille, Marseille, France.|Aix-Marseille Université, UM105, Marseille, France.
Jacques A
JA
Nunès
D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D017493:Leukocyte Common Antigens / Q000235:genetics*; D018950:Lipopolysaccharide Receptors / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics; D050799:STAT5 Transcription Factor / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects
D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D010750:Phosphoproteins; D050799:STAT5 Transcription Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D051057:Proto-Oncogene Proteins c-akt; D048052:Mitogen-Activated Protein Kinase 3; D017493:Leukocyte Common Antigens
cell signaling; flow cytometry; monocytes; phosphoflow analysis; single cell analysis