Human peripheral reticulocyte isolation and exosome release in vitro
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The exosomes released by peripheral reticulocytes were originally thought to function as vehicles for protein clearance for the maturing cells. With the emergence of exosomes as mediators of intercellular communication, a new paradigm exists for the role of reticulocyte-derived exosomes in both healthy and disease states, particularly conditions whose pathology is driven by the red blood cell and its precursors. However, no standard or detailed method for the isolation of human peripheral CD71+ reticulocytes exists. A combination of density-dependent and immunomagnetic approaches was used to demonstrate a procedure to isolate human CD71+ reticulocytes from peripheral blood. Nearly 90% of the CD71+ cells were distinct from the CD71- population when measured with flow cytometry detection of RNA. CD71+ reticulocyte-derived exosomes were then isolated and analyzed after incubation in vitro, the first such demonstration of these phenomena using these cells. These findings form the basis for more targeted and mechanistic studies into the role of reticulocyte-derived exosomes in pathologies like sickle cell disease.