RRML - Alternative Fixation Method Improves Flow Cytometry - Assisted Phospho-Detection Competence

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Concept, Design & Programming
Dr. Adrian Man

Nr. 18(4)/2010

Alternative Fixation Method Improves Flow Cytometry - Assisted Phospho-Detection Competence

Mihaela Zlei, Georgiana Grigore, Dragoş Peptanariu, Daniela Constantinescu, Corina Cianga, Eugen Carasevici, Monika Engelhardt


Analysis of protein phosphorylation within intracellular signaling pathways may clarify the functional status and biological responses of cells to various stimuli, reflecting an enhanced enzymatic activity. Phospho-specific antibody (p-Ab)-based detection methods that have been used to date, including flow cytometry, require extensive optimization for the achievement of accuracy and rational specificity. Objective. We sought to optimize a phospho-specific flow cytometry detection method, based on the premise that fixation is a critical step of such protocols. Materials and methods. We compared two different methods for phospho-detection in phorbol myristate acetate (PMA)-activated peripheral blood mononuclear cells (PBMCs) and assessed how they distinctly impacted on cell viability, surface staining intensity and phospho-detection competence, aiming to develop an optimized protocol with balanced efficiency in surface and intracellular phospho-labeling. PMA-activated PBMCs were used for testing the detection efficiency of five p-Abs: pERK(T202/Y204), pp38MAPK(T180/Y182), pAKT(T308), pSTAT-3(S727), pSTAT-1(Y701) by flow cytometry. Results. 1. Fixation prior to surface staining led to an improved signal-to-noise ratio for all p-Abs evaluated; 2. cell suspension integrity was not affected by the fixation method used; 3. reduced efficiency of surface marker detection levels remained within acceptable ranges; 4. one freeze-thaw cycle did not significantly impair the cell suspension integrity or staining efficiency. Conclusion. These results provide a practical approach for phospho-detection in heterogeneous tissue samples. The clinical relevance of our technical approach appears substantial, as frequent dysfunctions within signaling pathways are linked to various diseases and consequently require proper identification methodology.

Keywords: phosphorylation,cell signaling,flow cytometry

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How to cite
Zlei M, Grigore G, Peptanariu D, Constantinescu D, Cianga C, Carasevici E, et al. Alternative Fixation Method Improves Flow Cytometry - Assisted Phospho-Detection Competence. Rev Romana Med Lab. 2010;18(4):55-65