A simple new method for negative enrichment of monocytes from mouse blood and bone marrow (134.39)

Abstract

Abstract The mononuclear phagocyte system is comprised of tissue macrophages, dendritic cells, blood monocytes and their bone marrow (BM) progenitors. Transgenic mouse models have recently provided insight into the biology of monocytes in vivo. However a general method for isolating monocytes from mice is needed to better study their functions. We describe a rapid and simple method for the enrichment of monocytes from mouse BM and peripheral blood that does not require a density gradient and yields high purity and recovery. BM was harvested from femurs and tibia by crushing the bones. Blood was collected with heparin and red blood cells were removed by ammonium chloride lysis. The monocytes were then enriched using immunomagnetic, column-free negative selection (EasySep®). Briefly, unwanted cells were specifically labeled with dextran-coated magnetic particles using a cocktail of bispecific tetrameric antibody complexes. The sample was placed in a magnet and the supernatant containing unlabeled monocytes was collected. The separation procedure can be automated with a pipetting robot (RoboSep®). Purity of CD11b+Ly6G- cells as assessed by flow cytometry ranged from 80-93% for BM and 92-98% for blood with recovery of 46 ±11 % (n=38) and 25 ±10 % (n=20) respectively. This protocol will provide easy access to monocytes, enriched from peripheral blood and BM, for further studies of immune and inflammatory responses.