Detection of intracellular antigens in porcine PBMC by flow cytometry: A comparison of fixation and permeabilisation reagents.
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.
Gerner
Wilhelm
W
Clinical Immunology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria. wilhelm.gerner@vu-wien.ac.at
Käser
Tobias
T
Pintaric
Masa
M
Groiss
Sandra
S
Saalmüller
Armin
A
D000818:Animals; D051925:CD79 Antigens / Q000097:blood / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary*; D007371:Interferon-gamma / Q000097:blood / Q000276:immunology; D019394:Ki-67 Antigen / Q000097:blood / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000276:immunology*; D054353:Perforin / Q000097:blood / Q000276:immunology; D013552:Swine / Q000097:blood / Q000276:immunology*; D016707:Tissue Fixation / Q000379:methods / Q000662:veterinary
D051925:CD79 Antigens; D019394:Ki-67 Antigen; D054353:Perforin; D007371:Interferon-gamma