Gerner Wilhelm, Käser Tobias, Pintaric Masa, Groiss Sandra, Saalmüller Armin
Clinical Immunology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria.
Vet Immunol Immunopathol. 2008 Feb 15;121(3-4):251-9. doi: 10.1016/j.vetimm.2007.09.019. Epub 2007 Oct 2.
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.
细胞内抗原染色是流式细胞术的一种标准方法,但在兽医物种的免疫学研究中仍只是偶尔使用。为了获取更多关于猪淋巴细胞合适固定/通透方案的信息,我们测试了六种固定/通透变体,用于不同细胞内抗原的染色:CD79α、穿孔素、干扰素-γ和细胞周期相关蛋白Ki-67。对于固定/通透程序,我们测试了BD Biosciences、ADG Bio Research、Dako和AbD Serotec的四种商用试剂盒,并与甲醛非商用固定溶液以及用于通透的皂苷或吐温-20进行了比较。仅使用含皂苷的通透试剂(包括BD试剂盒)能最佳地染色表达穿孔素和Ki-67的细胞。相比之下,所有研究的固定/通透变体都能对CD79α和IFN-γ进行标记;然而,在此情况下,皂苷和吐温方案会诱导更高程度的非特异性结合。此外,与用ADG、Dako和Serotec试剂盒制备的样本相比用皂苷处理的细胞散射特性更差。细胞表面抗原的同时染色不受任何固定/通透变体的负面影响。我们的数据无法得出单一固定/通透策略的通用建议,但我们的工作为猪淋巴细胞中常见细胞内抗原的最佳染色提供了有用的指导方针。