Neutrophil Isolation

The purpose of this experiment was to utilize multiple methods to isolate splenic murine neutrophils and compare and contrast the effectiveness of these protocols based on 3 parameters: purity, viability and activation. We expected that each method would yield a distinct number of viable/pure neutrophils and that each isolation method would result in different degrees of activation of the neutrophils.
Conclusion:
In this study, we describe a modified magnetic bead isolation protocol for enriching spleen resident neutrophils by negative selection. EasySep® and Ficoll isolation methods fail to effectively purify neutrophils and particularly, in the case of Ficoll, yield a population of cells with low viability. While sort-purification generates a highly pure and viable population of neutrophils, the process of sorting leads to a substantial activation of the cells. Our results demonstrate that the OAC protocol yields a pure population of neutrophils. Equally important, the OAC protocol does not adversely affect cell viability or activation. Antibodies were titrated to determine optimal staining conditions. Gates were set based on previously determined FMO.