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Type of publication: Journal Article
Type of document: Full Paper

Year: 2008

Authors: Gerner, W; Käser, T; Pintaric, M; Groiss, S; Saalmüller, A

Title: Detection of intracellular antigens in porcine PBMC by flow cytometry: A comparison of fixation and permeabilisation reagents.

Source: Vet Immunol Immunopathol. 2008; 121(3-4):251-259

Authors Vetmeduni Vienna:

Gerner Wilhelm
Käser Tobias
Saalmüller Armin

Vetmed Research Units
Institute of Immunology

The staining of intracellular antigens is a standard method in flow cytometry but still only occasionally used for immunologic studies in veterinary species. In order to improve information about suitable fixation/permeabilisation protocols in porcine lymphocytes we tested six fixation/permeabilisation variants for the staining of different intracellular antigens: CD79alpha, perforin, interferon-gamma and the cell cycle associated protein Ki-67. For the fixation/permeabilisation procedure, four commercial kits from BD Biosciences, ADG Bio Research, Dako and AbD Serotec were tested in comparison to non-commercial fixation solutions of formaldehyde together with either saponin or Tween-20 for permeabilisation. Perforin and Ki-67 expressing cells were optimally stained only with permeabilisation reagents containing saponin, which includes the BD kit. In contrast, labelling of CD79alpha and IFN-gamma was possible with all investigated fixation/permeabilisation variants; however, here saponin and Tween protocols induced a higher degree of unspecific binding. Also, scatter properties of cells treated with saponin were worse compared to samples prepared with the kits from ADG, Dako and Serotec. Simultaneous staining of cell surface antigens was not negatively affected by any of the fixation/permeabilisation variants. A universal recommendation for a single fixation/permeabilisation strategy could not be deduced from our data but our work provides a useful guideline for optimal staining of common intracellular antigens analyzed in swine lymphocytes.

Keywords Pubmed: Animals
Antigens, CD79/blood
Antigens, CD79/immunology
Flow Cytometry/methods
Flow Cytometry/veterinary*
Ki-67 Antigen/blood
Ki-67 Antigen/immunology
Leukocytes, Mononuclear/chemistry
Leukocytes, Mononuclear/immunology*
Tissue Fixation/methods
Tissue Fixation/veterinary

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