A Gel-Based Dual Antibody Capture and Detection Method for Assaying of Extracellular Cytokine Secretion: EliCell

Abstract

SummaryA 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 antibodycontaining 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 fluorochromeconjugated antibody. Cytokine release and cellular morphology are visualized in parallel by phase contrast and fluorescence microscopy, respectively.

Leukocyte lipid bodies: inflammation-related organelles are rapidly detected by wet scanning electron microscopy

“…Staining artifacts were also observed. These have arisen from cellular autofluorescence exhibited by damaged or dying cells (z5% of the cells), as observed previously in other approaches using fluorescent products (25). Quantitative analyses of lipid bodies stained with osmium showed an increased number of these organelles in eotaxin-stimulated cells [3.7 6 1.5 lipid Fig.…”

“…Cell preparation was done in two ways: using a cytocentrifuge (17) or by spreading a mixture of eosinophils with melted agarose matrix (Pierce, Rockford, IL) onto a slide (25). OsO 4 staining and lipid body enumeration were performed exactly as described (17) (3 min), and then dried and mounted.…”