Spencer Lisa A, Melo Rossana C N, Perez Sandra A C, Weller Peter F
Department of Medicine, Harvard Thorndike Laboratories, Charles A. Dana Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
Methods Mol Biol. 2005;302:297-314. doi: 10.1385/1-59259-903-6:297.
A distinguishing feature of eosinophils is their ability to rapidly release preformed cytokines from intracellular pools. Cytokines are delivered to the cell surface from granule stores by transport vesicles and are released in small packets at discrete locations along the cell surface through a process termed "piecemeal" degranulation. The study of this process has been hindered by lack of an assay sensitive enough to register minute protein concentrations and the inability to visualize morphology of cytokine secreting cells. These hindrances have necessitated our development of the EliCell assay, an agarose-based dual cytokine capture and detection system through which cytokine secretion and cellular morphology may be analyzed in concert. Cells are embedded within capture antibody-containing agarose and stimulated under conditions of interest. Extracellularly released cytokine is captured within the matrix at the point of release from the cell and can be labeled with a fluorochrome-conjugated antibody. Cytokine release and cellular morphology are visualized in parallel by phase contrast and fluorescence microscopy, respectively.
嗜酸性粒细胞的一个显著特征是它们能够迅速从细胞内池释放预先形成的细胞因子。细胞因子通过运输囊泡从颗粒储存库输送到细胞表面，并通过一个称为“逐片”脱颗粒的过程在细胞表面的离散位置以小包形式释放。由于缺乏足够灵敏的检测方法来记录微小的蛋白质浓度，以及无法观察细胞因子分泌细胞的形态，对这一过程的研究受到了阻碍。这些障碍促使我们开发了EliCell检测法，这是一种基于琼脂糖的双细胞因子捕获和检测系统，通过该系统可以协同分析细胞因子分泌和细胞形态。将细胞包埋在含有捕获抗体的琼脂糖中，并在感兴趣的条件下进行刺激。细胞外释放的细胞因子在从细胞释放的点被捕获在基质中，并可以用荧光染料偶联抗体进行标记。分别通过相差显微镜和荧光显微镜并行观察细胞因子释放和细胞形态。
