EliCell assay for the detection of released cytokines from eosinophils

Journal of Leukocyte Biology

Spencer

Dvorak

et al. 2007

106

113

Eosinophils generate and store a battery of proteins, including classical cationic proteins, cytokines, chemokines, and growth factors. Rapid secretion of these active mediators by eosinophils is central to a range of inflammatory and immunoregulatory responses. Eosinophil products are packaged within a dominant population of cytoplasmic specific granules and generally secreted by piecemeal degranulation, a process mediated by transport vesicles. Large, pleiomorphic vesiculotubular carriers were identified recently as key players for moving eosinophil proteins from granules to the plasma membrane for extracellular release. During secretion, these specialized, morphologically distinct carriers, termed eosinophil sombrero vesicles, are actively formed and direct differential and rapid release of eosinophil proteins. This review highlights recent discoveries concerning the organization of the human eosinophil secretory pathway. These discoveries are defining a broader role for large vesiculotubular carriers in the intracellular trafficking and secretion of proteins, including selective receptor-mediated mobilization and transport of cytokines.

Section: Distinct Vesicular Compartments Operate In the Eosinophil Sementioning

confidence: 99%

Mechanisms of eosinophil secretion: large vesiculotubular carriers mediate transport and release of granule-derived cytokines and other proteins

Journal of Leukocyte Biology

Spencer

Dvorak

et al. 2007

106

113

“…Cell viability after stimulation was .95%, as determined by ethidium bromide incorporation. A nonphysiological challenge with the calcium ionophore A23187 (0.5 mM) was also used in some experiments, as described (22). Alternatively, unstimulated eosinophils were studied in whole samples of white blood cells isolated from human blood.…”

Section: Eosinophil Isolation Stimulation and Viabilitymentioning

confidence: 99%

“…A and B show cytospin and agarose cell preparations, respectively. Eosinophils were obtained from the blood of normal human donors and stimulated with eotaxin (A, B), calcium ionophore (C), or medium alone (D) as described previously (22). Fig.…”

Section: Light Microscopymentioning

confidence: 99%

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

Journal of Lipid Research

Sabban²,

Weller

2006

Leukocyte lipid bodies are dynamic, functionally active organelles with central roles in inflammation. Here, we report that leukocyte lipid bodies are facilely detected by a versatile, potent technique, termed wet scanning electron microscopy (SEM), which combines the rapid preparation of light microscopy with the resolution of SEM. Using as leukocyte models resting and agonist-stimulated human eosinophils, cells that generate prominent numbers of lipid bodies in inflammatory conditions, we demonstrated that lipid bodies can be rapidly imaged as bright, highly contrasted structures under wet SEM and scored by computerized image processing. Critical advantages of this approach are that it permits cell observation in a fully hydrated system and facilitates lipid preservation. These attributes are especially important because lipid bodies are degraded during routine dehydration processes. Moreover, this technology is advantageous over lipophilic fluorescent probes because it allows sustained detection of lipid bodies in contrast to short-lived fluorescent labeling of these organelles. The value of wet SEM in enabling rapid and largescale lipid body imaging and scoring within leukocytes is particularly important because lipid bodies are organelles underlying the heightened functions of inflammatory cells. Wet SEM technology provides new approaches and opportunities for delineations of lipid bodies in inflammatory diseases, including allergic inflammation.-Melo, R. C. N., A. Sabban, and P. F. Weller. Leukocyte lipid bodies: inflammation-related organelles are rapidly detected by wet scanning electron microscopy.

“…3A). A more diffuse staining pattern may indicate an insufficient concentration of capture antibody within the agarose matrix (18). In contrast, detection of products released through cytolysis (i.e., following stimulation with a calcium ionophore) will appear as a much more robust staining, without discrete limits (Fig.…”

Section: Positive Staining Results-mentioning

confidence: 99%

“…Released cytokine is captured within the matrix at the point of release from the cell and can be detected by a fluorochrome-labeled detection antibody. Viable cells remain embedded within the agarose substrate throughout the procedure and can be visualized under high magnification by phase contrast for morphological analysis in parallel with detection of released product by fluorescence microscopy (17,18).…”

Section: Introductionmentioning

confidence: 99%

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

Spencer

Perez

et al.

Handbook of ELISPOT

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.