de Caestecker M P, Telfer B A, Hutchinson I V, Ballardie F W
Department of Medicine, Manchester Royal Infirmary and University, UK.
J Immunol Methods. 1992 Sep 18;154(1):11-20. doi: 10.1016/0022-1759(92)90207-a.
Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.
采用双色流式细胞术分析人单核细胞原位细胞因子表达情况。将全血置于硅化玻璃瓶中培养，添加或不添加大肠杆菌脂多糖（LPS），培养不同时间，然后在进行流式细胞术分析前，使单核细胞（MNCs）经历多种通透处理程序。多聚甲醛（PF）/皂素固定可保留细胞形态，并产生可重复的通透程度（通过碘化丙啶掺入估计：平均94%，范围86 - 99%（n = 33））。在含有20%人血清的PBS中于0℃用4% PF固定并用1%皂素通透后，MNCs与藻红蛋白（PE）偶联的小鼠抗CD14（单核细胞表型）以及多克隆兔抗人白细胞介素 - 1α（IL - 1α）、IL - 1β、肿瘤坏死因子α（TNF - α）或对照兔IgG一起孵育。使用山羊抗兔IgG异硫氰酸荧光素（FITC）检测兔抗体的结合情况。与对照相比，LPS刺激后，三种抗细胞因子抗体使CD14 PE阳性细胞中的FITC荧光增强。LPS刺激后，单核细胞IL - 1β和TNF - α表达呈现可重复的剂量相关反应，TNF - α（2小时）出现早期峰值，而IL - 1β（4小时）和IL - 1α（12小时）出现峰值时间较晚。使用相应的重组人细胞因子进行抑制研究、CD14阴性或未通透的MNCs中无染色以及通过紫外线免疫化学观察到不同细胞因子的特征性细胞质定位，证实了该细胞因子检测系统的特异性。因此，本文所述方法为检测细胞相关单核因子提供了一种可重复、半定量且特异的检测方法。该技术可能适用于分析其他表型明确的细胞群体中的多种不同细胞因子。
