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

ISSN online: 2284-5623

ISSN-L: 1841-6624

Rejection rate (2020): 75%

Română English

Journal Metrics

Impact Factor 0.5
Five Year Impact Factor 0.5
JCI 0.12

Advanced search

Top 10 downloaded articles
- April 2024 -
 
A comprehensive review of Prof... 25
Recomandarea comună EFLM-COLA... 15
Anti-thyroid peroxidase (TPO) ... 11
Understanding the key differen... 10
Monocyte to high-density lipop... 10
Romanian Review of Laboratory ... 7
Understanding the pathogenesis... 7
Predictive value of expression... 7
Approaching Risk Management in... 6
Function of the S1P pathway in... 6

Log in

Concept, Design & Programming
Dr. Adrian Man
   
 
Nr. 18(4)/2010
XML
TXT

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

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


Abstract:

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

 
  PDF Download full text PDF
(391 KB)
     
 
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