A rapid method for the isolation of Eosinophils without Ficoll or RBC Lysis (38.1)

Abstract

Abstract Eosinophils typically comprise 1–5% of blood leucocytes in non-allergic, healthy humans. Allergic and asthmatic reactions result in activation of eosinophils with resultant release of cytoplasmic granules, cytokines and lytic enzymes. Eosinophils are also mediators of responses against parasitic infections. Many protocols for their isolation stress the avoidance of ammonium chloride lysis as this interferes with eosinophil antigen processing, cytokine responses and alters cell morphology. We describe a rapid and simple method for the enrichment of eosinophils from normal blood that does not require Ficoll or lysis steps and yields good purity and recovery. Blood was collected with heparin and RBC were removed by HetaSep sedimention. The eosinophils were then enriched using immunomagnetic, column-free negative selection (EasySep®). Briefly, cells were labeled with a cocktail of antibodies targeting unwanted cells. These were then coupled to magnetic nanoparticles and the sample was placed in a magnet. The labeled cells were thus removed leaving unlabeled eosinophils. The entire separation procedure can be automated with a pipetting robot (RoboSep®). Purity of eosinophils assessed by cytospin and Wright’s stain was 98–99%. By flow cytometry (FACS) eosinophils were defined as CD45+CD66b+ and CD16 negative with low side scatter. Final purity assessed by FACS was 94 ± 3%. Recovery averaged 69 ± 21 (n=11).