Separation of bovine bone marrow into maturation-related myeloid cell fractions

A prerequisite for studies on bovine myeloid cells in relation to maturity is a reliable separation method, in order to obtain enriched and partially purified cell fractions of different maturation stages. Since current techniques for bovine bone marrow cell isolation fall short of this requirement, a technique for fractionating bovine bone marrow using a three-layer discontinuous Percoll gradient was developed. Three maturation-dependent myeloid cell fractions were obtained at specific densities, as maturation of cells is accompanied with a progressive density increase. Early immature myeloid cells, i.e. myeloblasts and promyelocytes, were found at a density of 1.060 g/ml. Late immature myeloid cells, i.e. myelocytes and metamyelocytes, were retrieved at 1.080 g/ml. Bands and segmented cells, representing the mature fraction, accumulated in the high-density pellet (>1.080 g/ml). Myeloid cell populations were identified in each fraction by flow cytometry based on their forward and side scatter pattern. Confirmation was provided by light microscopy of flow cytometrically sorted myeloid populations, using morphological characteristics. The developed method provides a unique tool for studying maturation-dependent functions in bovine bone marrow.