Prussin C, Metcalfe D D
Allergic Diseases Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-1888, USA.
J Immunol Methods. 1995 Dec 15;188(1):117-28. doi: 10.1016/0022-1759(95)00209-x.
Recently, there have been several reports demonstrating improvements in the flow cytometric detection of intracellular cytokines. These advances, although significant, have not yielded techniques that have easily been translated into broad use. To address this issue, we have coupled a fixation and permeabilization method with the use of directly labelled monoclonal anti-cytokine antibodies, providing both improved signal and simpler staining. The kinetics of in situ cytokine production in both CD4 and CD8 cells are shown for IL-2, IL-4, IL-5 and IFN-gamma. Based on these data, 6 h was chosen for optimal detection of this combination of cytokines. We show the specificity of this technique by blocking cytokine staining using a molar excess of recombinant cytokine. Additionally, unlabelled anti-cytokine antibodies are demonstrated to block specific staining of labelled antibody, providing an objective means to place statistical markers. Using such controls, we routinely detected as few as 0.1% false positive cells, allowing the flow cytometric detection of IL-5, which is below the threshold of detection of published methods. To further prove the specificity of staining, we stained using two anti-IL-5 mAbs known to recognize different epitopes and demonstrate that the same cells stain with both antibodies. Without permeabilization we could detect a fraction of cells with low intensity staining for cytokine. This staining was further examined using differential two color staining for intracellular and extracellular cytokine, clearly demonstrating no cells staining exclusively for extracellular cytokine, confirming a lack of passive transfer of cytokine to nearby cells. We show that cytokine flow cytometry is useful in examining the increased IL-5 production characteristic of eosinophilic states and that IL-5 production is limited to the CD27 negative subpopulation. These data illustrate the unique capability of cytokine flow cytometry to correlate cytokine expression with cell surface phenotype without cell separation. In summary, using directly conjugated anti-cytokine antibodies, cytokine flow cytometry becomes a specific and versatile technique for the assessment of complex cytokine production phenotypes in fresh ex vivo T cell subpopulations.
最近，有几份报告表明细胞内细胞因子的流式细胞术检测有了改进。这些进展虽然显著，但尚未产生易于广泛应用的技术。为了解决这个问题，我们将固定和通透化方法与直接标记的抗细胞因子单克隆抗体的使用相结合，既提高了信号又简化了染色。展示了IL-2、IL-4、IL-5和IFN-γ在CD4和CD8细胞中原位细胞因子产生的动力学。基于这些数据，选择6小时用于最佳检测这种细胞因子组合。我们通过使用摩尔过量的重组细胞因子阻断细胞因子染色来展示该技术的特异性。此外，未标记的抗细胞因子抗体被证明可阻断标记抗体的特异性染色，提供了一种放置统计标记的客观方法。使用这样的对照，我们常规检测到低至0.1%的假阳性细胞，从而能够对流式细胞术检测IL-5，其低于已发表方法的检测阈值。为了进一步证明染色的特异性，我们使用两种已知识别不同表位的抗IL-5单克隆抗体进行染色，并证明相同的细胞用两种抗体都能染色。未经通透化处理时，我们可以检测到一小部分细胞对细胞因子有低强度染色。使用细胞内和细胞外细胞因子的差异双色染色对这种染色进行了进一步检查，清楚地表明没有细胞仅对细胞外细胞因子染色，证实细胞因子没有被动转移到附近细胞。我们表明细胞因子流式细胞术可用于检查嗜酸性粒细胞状态特有的IL-5产生增加情况，并且IL-5产生仅限于CD27阴性亚群。这些数据说明了细胞因子流式细胞术在不进行细胞分离的情况下将细胞因子表达与细胞表面表型相关联的独特能力。总之，使用直接偶联的抗细胞因子抗体，细胞因子流式细胞术成为一种用于评估新鲜离体T细胞亚群中复杂细胞因子产生表型的特异性和通用技术。
