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MicroRNA profiling in human neutrophils during bone marrow granulopoiesis and in vivo exudation.

PLoS One

September 2013

The Granulocyte Research Laboratory, Department of Hematology, National University Hospital, University of Copenhagen, Copenhagen, Denmark.

The purpose of this study was to describe the microRNA (miRNA) expression profiles of neutrophils and their precursors from the initiation of granulopoiesis in the bone marrow to extravasation and accumulation in skin windows. We analyzed three different cell populations from human bone marrow, polymorphonuclear neutrophil (PMNs) from peripheral blood, and extravasated PMNs from skin windows using the Affymetrix 2.0 platform.

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To evaluate the mechanism of neutrophilia by granulocyte colony-stimulating factor (G-CSF), kinetic studies with tritiated thymidine ([3H]TdR) were performed in mice. G-CSF increased the number of circulating metamyelocytes and band and segmented neutrophils after the daily injection of G-CSF. The production of neutrophils has been estimated from the number of postmitotic neutrophils and the marrow transit time of [3H]TdR-labeled neutrophils on day 4 of daily G-CSF injection.

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The soft agar culture system is widely used to study in vitro regulation of granulopoiesis. This report is presented to illustrate how agar itself affects the kinetics of colony growth and thus, the results obtained with the assay. The main finding was that agar caused cells to lyse as they matured.

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Fifty-three maturing bone marrow cells of the granulocyte cell series stained with Giemsa stain and magnified 1,000 times were scanned by a "computerized microscope" consisting of a LSI-11/23 microprocessor and a black-and-white video camera attached to a "frame grabber ." Each sampled cell was digitized into 70 X 70 pixels, each pixel representing 0.04 micron of the real image.

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