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Mediators of Inflammation
Volume 2014, Article ID 418292, 12 pages
http://dx.doi.org/10.1155/2014/418292
Research Article

5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity

1Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Martinistrasße 52, 20246 Hamburg, Germany
2Department of Oncology/Hematology, University Medical Center Hamburg-Eppendorf, Martinistrasße 52, 20246 Hamburg, Germany
3Department for Transfusion Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasße 52, 20246 Hamburg, Germany

Received 6 December 2013; Accepted 28 January 2014; Published 13 March 2014

Academic Editor: Beatrice Gaugler

Copyright © 2014 Thomas Stübig et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Flow cytometric analysis of intracellular granzyme and analysis of naïve cells T-cells were purified and kept as described. T-cells were treated with the mentioned concentrations of 5-Aza.

For the analysis of intracellular proteins T- cells were stimulated with phorbol myristate acetate (PMA), Ionomycin for 1 hour and Brefeldin A for 4.5 hours. All chemicals were obtained from Sigma-Aldrich (Munich, Germany). Cells were harvested and prepared for analysis using the Cytofix/Cytoperm kit (BD Bioscience, Heidelberg, Germany). For intracellular cell staining the following antibody was used: anti-Granzyme-PE. Prior to intracellular staining cells were marked with anti-CD4-FITC, and anti-CD8-APC (all antibodies obtained from BD Bioscience, Heidelberg, Germany).

For analysis of naïve T-cells, cells were stained after Aza treatment with the following antibodies: anti-CD4- PerCP, anti-CD8-APC, anti-CCR7-PE, and anti- CD45RA-FITC (all antibodies obtained from BD Bioscience, Heidelberg, Germany).

All analysis were performed using a Canto II (BD Bioscience, Heidelberg, Germany) and data were further analysed using the FlowJo Software (TreeStar Inc, Ashland, USA).

  1. Supplementary Material