Format

Send to

Choose Destination
J Immunol Methods. 2014 Dec 15;415:46-56. doi: 10.1016/j.jim.2014.09.003. Epub 2014 Sep 28.

Rapid and reliable determination of the halogenating peroxidase activity in blood samples.

Author information

1
Translational Centre for Regenerative Medicine (TRM) Leipzig, Philipp-Rosenthal-Straße 55, 04103 Leipzig, Germany; Institute for Medical Physics and Biophysics, Medical Faculty, University of Leipzig, Härtelstraße 16-18, 04107 Leipzig, Germany. Electronic address: joerg.flemmig@uni-leipzig.de.
2
Institute for Medical Physics and Biophysics, Medical Faculty, University of Leipzig, Härtelstraße 16-18, 04107 Leipzig, Germany. Electronic address: pauline_schwarz@web.de.
3
Fraunhofer Institute for Cell Therapy and Immunology (IZI) Leipzig, Perlickstraße 1, 04103 Leipzig, Germany. Electronic address: ingo.baecker@izi.fraunhofer.de.
4
Fraunhofer Institute for Cell Therapy and Immunology (IZI) Leipzig, Perlickstraße 1, 04103 Leipzig, Germany. Electronic address: anna.leichsenring@izi.fraunhofer.de.
5
Fraunhofer Institute for Cell Therapy and Immunology (IZI) Leipzig, Perlickstraße 1, 04103 Leipzig, Germany. Electronic address: franziska.lange@izi.fraunhofer.de.
6
Translational Centre for Regenerative Medicine (TRM) Leipzig, Philipp-Rosenthal-Straße 55, 04103 Leipzig, Germany; Institute for Medical Physics and Biophysics, Medical Faculty, University of Leipzig, Härtelstraße 16-18, 04107 Leipzig, Germany. Electronic address: juergen.arnhold@medizin.uni-leipzig.de.

Abstract

By combining easy and fast leukocyte enrichment with aminophenyl-fluorescein (APF) staining we developed a method to quickly and specifically address the halogenating activity of the immunological relevant blood heme peroxidases myeloperoxidase and eosinophil peroxidase, respectively. For leukocyte enrichment a two-fold hypotonic lysis procedure of the blood with Millipore water was chosen which represents a cheap, fast and reliable method to diminish the amount of erythrocytes in the samples. This procedure is shown to be suitable both to human and murine blood micro-samples, making it also applicable to small animal experiments with recurring blood sampling. As all types of leukocytes are kept in the sample during the preparation, they can be analysed separately after discrimination during the flow cytometry analysis. This also holds for all heme peroxidase-containing cells, namely neutrophils, eosinophils and monocytes. Moreover additional parameters (e.g. antibody staining) can be combined with the heme peroxidase activity determination to gain additional information about the different immune cell types. Based on previous results we applied APF for specifically addressing the halogenating activity of leukocyte peroxidases in blood samples. This dye is selectively oxidized by the MPO and EPO halogenation products hypochlorous and hypobromous acid. This approach may provide a suitable tool to gain more insights into the immune-physiological role of the halogenating activity of heme peroxidases.

KEYWORDS:

Aminophenyl fluorescein; Blood preparation; Chlorinating activity; Myeloperoxidase; Neutrophils

PMID:
25264081
DOI:
10.1016/j.jim.2014.09.003
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center