Zeller J M, Rothberg L, Landay A L
Department of Medical Nursing, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL 60612.
Clin Exp Immunol. 1989 Oct;78(1):91-6.
Assays routinely employed to evaluate human monocyte respiratory burst activation have been limited to measuring responses of bulk cell preparations. We demonstrate that individual monocyte responses can be easily assessed by using 2',5' dichlorofluorescin diacetate (DCFH-DA) and flow cytometry. Adherence purified monocytes were incubated with DCFH-DA, washed, and stimulated with either phorbol myristate acetate (PMA) or heat-aggregated IgG (HAIgG). Log green fluorescence signals were measured by using a flow cytometer equipped with a 5-W argon laser set at an excitation wavelength of 488 nm. Optimal conditions for stimulation included exposure to 5 microM concentrations of DCFH-DA for 15 min, followed by a 60-min incubation with either PMA or HAIgG. Dichlorofluorescin (DCFH) oxidation by monocytes increased in a graded fashion as a function of stimulus concentration. Monocytes responded as a uniform population in response to increasing doses of PMA and HAIgG. This oxidative response was also monitored in mixed populations of mononuclear leukocytes, with monocytes identified on the basis of light scatter properties and surface antigen staining with anti-CD14. More than 90% of cells demonstrating increases in log green fluorescence signals following activation were CD14 positive. Measurement of DCFH oxidation by monocytes is reflective of the capacity to undergo a respiratory burst response, in that monocytes obtained from patients with chronic granulomatous disease were only minimally reactive. This assay, representing a rapid means of assessing monocyte respiratory burst activation by single cell analysis, is suitable for use in both clinical and research settings.
常规用于评估人类单核细胞呼吸爆发激活的检测方法一直局限于测量大量细胞制剂的反应。我们证明，使用二氯荧光素二乙酸酯（DCFH-DA）和流式细胞术可以轻松评估单个单核细胞的反应。将贴壁纯化的单核细胞与DCFH-DA孵育，洗涤，然后用佛波酯肉豆蔻酸酯（PMA）或热聚集IgG（HAIgG）刺激。使用配备5瓦氩离子激光的流式细胞仪在488 nm激发波长下测量对数绿色荧光信号。刺激的最佳条件包括暴露于5 microM浓度的DCFH-DA 15分钟，然后与PMA或HAIgG孵育60分钟。单核细胞对二氯荧光素（DCFH）的氧化随着刺激浓度的增加呈分级增加。单核细胞对增加剂量的PMA和HAIgG以均匀群体的形式做出反应。在单核白细胞的混合群体中也监测到这种氧化反应，单核细胞根据光散射特性和抗CD14表面抗原染色进行鉴定。激活后显示对数绿色荧光信号增加的细胞中，超过90%是CD14阳性。单核细胞对DCFH氧化的测量反映了其进行呼吸爆发反应的能力，因为从慢性肉芽肿病患者获得的单核细胞反应性极低。该检测方法代表了一种通过单细胞分析评估单核细胞呼吸爆发激活的快速方法，适用于临床和研究环境。
