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Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments
Published on: July 10, 2013
Purification and functional evaluation of mature neutrophils from human bone marrow
Abstract:
Human myeloid maturation proceeds within the bone marrow and results in a mature neutrophil that is released into the peripheral circulation. Previous reports have indicated that neutrophils from bone marrow demonstrate decreased adherence, impaired phagocytosis, and decreased nitroblue tetrazolium dye reduction when stimulated. Due to lack of a suitable method for isolating purified bone marrow neutrophils, these studies have been performed by microscopic techniques. We now report a method for isolating 1 X 10(8) neutrophils [bands plus polymorphonuclear leukocytes (PMNs)] from 10 mL of bone marrow aspirate sample. By means of a discontinuous Percoll-gradient centrifugation through densities of 1.085, 1.095, and 1.10 g/mL a leukocyte-rich suspension of bone marrow can be separated into three leukocyte layers. By combining the lower two leukocyte layers (M2/3), a population of neutrophils consisting of 26% bands and 63% PMNs is seen. When compared with peripheral blood PMNs, these bone marrow neutrophils had a lower alkaline phosphatase activity, decreased ingestion of Oil Red O-coated particles, impaired superoxide release on stimulation with the chemotactic peptide Fmet-leu-phe (FMLP) or the tumor promotor phorbol myristate acetate (PMA), and less activity of the NADPH-dependent oxidase. These results indicate that morphologically mature neutrophilic cells within the bone marrow exist in a still functionally immature state.
Insights
Bone marrow neutrophils, though morphologically mature, exhibit functional immaturity. A new isolation method reveals impaired adherence, phagocytosis, and reduced oxidative burst activity in these immature neutrophils.
Area of Science:
- Hematology
- Cell Biology
- Immunology
Background:
- Human myeloid maturation culminates in circulating neutrophils.
- Previous studies on bone marrow neutrophils were limited by isolation methods and microscopic techniques.
- Reported functional deficits in bone marrow neutrophils include decreased adherence and phagocytosis.
Purpose of the Study:
- To develop a method for isolating purified bone marrow neutrophils.
- To compare the functional characteristics of isolated bone marrow neutrophils with peripheral blood neutrophils.
Main Methods:
- Isolation of 1 x 10(8) neutrophils (bands and PMNs) from 10 mL bone marrow aspirate.
- Discontinuous Percoll-gradient centrifugation using densities of 1.085, 1.095, and 1.10 g/mL.
- Assessment of alkaline phosphatase activity, Oil Red O particle ingestion, superoxide release (FMLP/PMA stimulated), and NADPH oxidase activity.
Main Results:
- A neutrophil population (26% bands, 63% PMNs) was isolated using Percoll gradients.
- Bone marrow neutrophils exhibited lower alkaline phosphatase activity compared to peripheral PMNs.
- Impaired superoxide release and reduced NADPH oxidase activity were observed in bone marrow neutrophils.
Conclusions:
- Morphologically mature neutrophils in bone marrow are functionally immature.
- The developed isolation method enables detailed functional analysis of bone marrow neutrophils.
- These findings highlight distinct functional states between bone marrow and circulating neutrophils.
