Sewell W A, North M E, Webster A D, Farrant J
MRC Immunodeficiency Research Group, Department of Clinical Immunology, Royal Free Hospital School of Medicine, London, UK.
J Immunol Methods. 1997 Nov 10;209(1):67-74. doi: 10.1016/s0022-1759(97)00150-6.
Various methods have been reported for measuring intracellular cytokines in peripheral blood mononuclear cells isolated by density-gradient centrifugation. In this report, we describe a whole-blood method for the determination of intracellular cytokines (IFN-gamma, TNF-alpha and IL-2) that uses small-volume (500 microl) blood samples. Directly conjugated anti-cytokine antibodies and commercial cell membrane fixation and permeabilisation reagents were used. Blood was cultured in a 1:3 dilution with a combination of PMA and ionomycin to reveal the cytokine synthetic potential of each cell, together with monensin to increase the sensitivity by retaining cytokines within the cell to detectable levels. The optimum concentrations of PMA (10 ng/ml (16.2 nmol/l)), ionomycin (2 micromol/l) and monensin (3 micromol/l) were determined. Kinetic studies showed maximal cytokine expression after 2 h of culture for TNF-alpha and IFN-gamma and 4 h for IL-2. Assessment of TNF-alpha and IFN-gamma production within the CD4 and CD8 lymphocytes from 10 normal volunteers showed that considerably more CD8 + than CD4 + cells produced IFN-gamma. This technique could be used by routine immunology laboratories and will be of use in studies to determine whether cytokine assays are of value in the investigation of immune disorders.
已有多种方法用于检测通过密度梯度离心分离的外周血单核细胞中的细胞内细胞因子。在本报告中，我们描述了一种用于测定细胞内细胞因子（IFN-γ、TNF-α和IL-2）的全血方法，该方法使用小体积（500微升）血样。使用了直接偶联的抗细胞因子抗体以及市售的细胞膜固定和通透试剂。将血液以1:3的稀释度与佛波酯（PMA）和离子霉素混合培养，以揭示每个细胞的细胞因子合成潜力，同时加入莫能菌素，通过将细胞因子保留在细胞内至可检测水平来提高灵敏度。确定了PMA（10 ng/ml（16.2 nmol/l））、离子霉素（2 μmol/l）和莫能菌素（3 μmol/l）的最佳浓度。动力学研究表明，培养2小时后TNF-α和IFN-γ的细胞因子表达达到最大值，IL-2则在4小时达到最大值。对10名正常志愿者的CD4和CD8淋巴细胞内TNF-α和IFN-γ产生情况的评估显示，产生IFN-γ的CD8 +细胞比CD4 +细胞多得多。该技术可供常规免疫学实验室使用，将有助于确定细胞因子检测在免疫紊乱研究中是否有价值的研究。
