Carey L A, Schuhl R A, Gee M H
Department of Physiology, Jefferson Medical College Thomas Jefferson University, Philadelphia, PA 19107, USA.
Biotechniques. 1995 Nov;19(5):824-9.
We report a microplate reader assay for the measurement of superoxide anion (O2-) production from opsonized zymosan-stimulated sheep polymorphonuclear leukocytes (PMNs). This method, for use with the particulate neutrophil activator, is a modification of the cytochrome c reduction system that measures O2- release from adherent neutrophils stimulated with soluble mediators such as phorbol-myristate-acetate (PMA). We measured O2- release from PMNs (250,000 per well) stimulated with opsonized zymosan over a dose range from 0.0625 mg/mL to 0.5 mg/mL. A dose of 0.125 mg/mL was shown to stimulate maximal superoxide production while having little effect on the optical density of the reagent blank. O2- values obtained using this method were not statistically different from values measured using cell-free supernatants. This system was useful in demonstrating PMN priming, as evidenced by the 2.7 times increase in O2- production above baseline from neutrophils isolated from sheep given a 12-hour, low-dose endotoxin infusion. This is a convenient and sensitive method for measurement of neutrophil O2- production in response to a receptor-mediated, particulate stimulus.
我们报告了一种微孔板读取器检测方法，用于测量经调理酵母聚糖刺激的绵羊多形核白细胞（PMN）产生超氧阴离子（O2-）的情况。这种用于颗粒性中性粒细胞激活剂的方法，是对细胞色素c还原系统的一种改进，该系统用于测量由佛波酯-肉豆蔻酸酯-乙酸酯（PMA）等可溶性介质刺激的贴壁中性粒细胞释放的O2-。我们测量了在0.0625 mg/mL至0.5 mg/mL剂量范围内，经调理酵母聚糖刺激的PMN（每孔250,000个）释放的O2-。结果显示，0.125 mg/mL的剂量可刺激产生最大量的超氧化物，同时对试剂空白的光密度影响很小。使用该方法获得的O2-值与使用无细胞上清液测量的值在统计学上无差异。该系统可用于证明PMN的预激活，从接受12小时低剂量内毒素输注的绵羊分离出的中性粒细胞产生的O2-比基线水平增加2.7倍，这一结果就证明了这一点。这是一种方便且灵敏的方法，用于测量中性粒细胞对受体介导的颗粒性刺激产生O2-的情况。
