Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Aug 27;12(1):110-20.
doi: 10.2203/dose-response.13-015.Silva. eCollection 2014 Jan.

Simultaneous analysis of p53 protein expression and cell proliferation in irradiated human lymphocytes by flow cytometry

Rafael de Freitas E Silva  1 , Neyliane Frassinetti Gonçalves Dos Santos  1 , Valéria Rěgo Alves Pereira  2 , Ademir Amaral  1
Affiliations

Simultaneous analysis of p53 protein expression and cell proliferation in irradiated human lymphocytes by flow cytometry

Rafael de Freitas E Silva et al. Dose Response. .

Abstract

P53 protein has an intrinsic role in modulating the cellular response against DNA radioinduced damages and has been pointed out as an indirect indicator of individual radiosensitivity. The rate of cell proliferation is also a parameter that has been related to tissue sensitivity to radiation. However, this feature is yet understudied. In this context, the aim of this work was to employ Flow Cytometry (FC) for simultaneously assessing of p53 protein expression levels together with cellular proliferation rate of irradiated human lymphocytes. From in vitro irradiated human blood samples, mononuclear cells were isolated and labeled with Carboxylfluorescein Diacetate Succinimidyl Ester (CFSE) prior to phytohaemagglutinin (PHA) stimulation in culture for 96 hours. Cells were also labeled with anti-p53 monoclonal antibody PE-conjugated in order to analyze either proliferation rate or p53 expression levels by FC. It was verified a reduction in the proliferation rate of irradiated lymphocytes and, in parallel, a rise in the p53 expression levels, similar for quiescent and proliferating lymphocytes. The results emphasize the importance of the use of CFSE-stained lymphocytes in assays associated to proliferation rate and the use of this methodology in several studies, such as for evaluating individual radiosensitivity.

Keywords: CFSE; Flow cytometry; P53 protein; Proliferation rate; Radiosensitivity.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Dot-plots obtained during analysis of lymphocytes non-stimulated with mitogen, non-irradiated (A) and irradiated with 4 Gy (B).The upper quadrants allows the visualization of p53 expression (left, negative; right, positive) and the lower quadrants, the pattern of proliferation (left, proliferating; right, quiescent).
FIG. 2.
FIG. 2.
Dot-plots obtained during the analysis of non-irradiated (0 Gy) and irradiated (4 Gy) proliferating samples from subjects A, B, C, D and E. Lower right quadrant displays quiescent lymphocytes (CFSE high fluorescence) non-positive for p53 and upper right quadrant displays quiescent cells positive for p53 expression; lower left quadrant displays proliferating lymphocytes (CFSE low fluorescence) non-positive for p53 and upper left quadrant displays proliferating cells positive for p53 expression.
See this image and copyright information in PMC

References

    1. Andrzej D, Elzbieta B, Wojciech G, Zbigniew D. Activation of nuclear factor kappa B (NF-kB) assayed by laser scanning cytometry (LSC) Cytometry. 1998;33:376–382. - PMC - PubMed
    1. Asquith B, Debacq C, Florins A, Gillet N, Sanchez-Alcaraz T, Mosley A, Willems L. Quantifying lymphocyte kinetics in vivo using carboxyfluoresceindiacetatesuccinimidyl ester (CFSE) Proc R Soc Lond B Biol Sci. 2006;273:1165–1171. - PMC - PubMed
    1. Banks HT, Sutton KL, Thompson WC, Bocharov G, Doumic M, Schenkel T, Argilaguet J, Giest S, Peligero C, Meyerhans A. A new model for the estimation of cell proliferation dynamics using CFSE data. J Immunol Methods. 2011;373:143–160. - PMC - PubMed
    1. Barnett GC, West CM, Dunning AM, Elliott RM, Coles CE, Pharoah PD, Burnet NG. Normal tissue reactions to radiotherapy: towards tailoring treatment dose by genotype. Nat Rev Cancer. 2009;9:134–142. - PMC - PubMed
    1. Borgmann K, Röper B, El-Awady R, Brackrock S, Bigalke M, Dörk T, Alberti W, Dikomey E, Dahm-Daphi J. Indicators of late normal tissue response after radiotherapy for head and neck cancer: fibroblasts, lymphocytes, genetics, DNA repair, and chromosome aberrations. Radiother Oncol. 2002;64:141–152. - PubMed

LinkOut - more resources