Gröger M, Matsumura T, Kohrgruber N, Maurer D, Wolff K, Petzelbauer P
Division of General Dermatology, University of Vienna Medical School, Austria.
J Immunol Methods. 1999 Jan 1;222(1-2):101-9. doi: 10.1016/s0022-1759(98)00187-2.
We describe an automated, observer-independent and highly reproducible assay for the quantification of transmigrated neutrophils across endothelial monolayers. Endothelial cells grown on collagen gels were loaded with a dye emitting red fluorescence. Neutrophils loaded with dye emitting green fluorescence were allowed to adhere to and transmigrate across endothelial monolayers. For quantification of adherent and migrated cells, randomly selected fields were scanned by confocal laser scan microscopy at defined depths within and below the endothelial monolayers. The images obtained were transferred into the public domain NIH image program and numbers and distribution of cells within scanned sectors were automatically calculated. We demonstrate that adherent neutrophils are easily discriminated from transmigrated cells; absolute numbers of migrated cells can be reproducibly calculated by counting cells at a depth of -20 microm, thus permitting evaluation of large-scale experiments: the efficacy of neutrophil transmigration depends on the level of endothelial activation after TNF stimulation and mAbs to cell surface adhesion molecules interfere with migration in a manner similar to that previously shown in in vivo experiments. This assay lends itself to the identification of molecules influencing in cell migration in each phase of EC activation and to the screening of pro- and anti-migratory properties of biological or pharmacological reagents.
我们描述了一种用于定量检测中性粒细胞穿过内皮细胞单层迁移情况的自动化、与观察者无关且高度可重复的检测方法。在胶原蛋白凝胶上生长的内皮细胞被加载一种发出红色荧光的染料。加载发出绿色荧光染料的中性粒细胞被允许黏附并穿过内皮细胞单层。为了定量检测黏附细胞和迁移细胞，在内皮细胞单层内部及下方的特定深度，通过共聚焦激光扫描显微镜对随机选择的视野进行扫描。获得的图像被传输到公共领域的美国国立卫生研究院图像程序中，并自动计算扫描区域内细胞的数量和分布。我们证明黏附的中性粒细胞很容易与迁移的细胞区分开来；通过在-20微米深度计数细胞，可以可重复地计算出迁移细胞的绝对数量，从而能够评估大规模实验：中性粒细胞迁移的效率取决于肿瘤坏死因子刺激后内皮细胞的活化水平，并且针对细胞表面黏附分子的单克隆抗体以类似于先前在体内实验中所显示的方式干扰迁移。这种检测方法有助于鉴定在内皮细胞活化的每个阶段影响细胞迁移的分子，并筛选生物或药理试剂的促迁移和抗迁移特性。
