Cell-free granules, upon extrusion from human eosinophils, remain fully competent to secrete granule-derived proteins in receptor-mediated processes in response to different stimuli. However, in order to avoid the shrinkage and damage of granules, as well as preserve their structure, properties, and functionality, the use of an optimized process of subcellular fractionation using an isoosmotic density gradient is needed. Here, we describe a detailed protocol of subcellular fractionation of nitrogen-cavitated eosinophils on an isoosmotic iodinated density gradient (iodixanol) and the isolation of well-preserved and functional membrane-bound specific granules.
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The authors thank Conselho Nacional de Pesquisas (CNPq—Brazil), Comissão de Aperfeiçoamento de Nível Superior (Capes—Brazil), and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ—Brazil) for the financial support.
Neves JS, Perez SA, Spencer LA, Melo RC, Reynolds L, Ghiran I et al (2008) Eosinophil granules function extracellularly as receptor-mediated secretory organelles. Proc Natl Acad Sci U S A 105:18478–18483PubMedCentralPubMedCrossRefGoogle Scholar
Neves JS, Radke AL, Weller PF (2010) Cysteinyl leukotrienes acting via granule membrane-expressed receptors elicit secretion from within cell-free human eosinophil granules. J Allergy Clin Immunol 125:477–482PubMedCentralPubMedCrossRefGoogle Scholar
Neves JS, Perez SA, Spencer LA, Melo RC, Weller PF (2009) Subcellular fractionation of human eosinophils: isolation of functional specific granules on isoosmotic density gradients. J Immunol Methods 344:64–72PubMedCentralPubMedCrossRefGoogle Scholar
Akuthota P, Shamri R, Weller PF (2012) Isolation of human eosinophils. In: Wiley, J. & Sons (ed) Curr Protocols Immunol 7: 31Google Scholar