Isolation and High Throughput Flow Cytometric Apoptosis Assay of Human Neutrophils to Enable Compound Library...

The study of human neutrophils in vitro is challenging due to their short half-life and propensity for activation. However, with careful handling and manipulation in the laboratory, they can be a powerful tool to investigate immune responses in health and disease. Here we describe a method for the isolation of human neutrophils from peripheral blood samples, followed by a high-throughput screen to assess the efficacy of a library of compounds in inducing neutrophil apoptosis, which may have therapeutic potential in neutrophil-driven diseases. This protocol is based on previously-published neutrophil isolation methods utilizing Dextran sedimentation of red blood cells followed by the separation of granulocytes with plasma/Percoll discontinuous gradient centrifugation. Yields of ~1 x 10⁶ neutrophils per millilitre of blood, and purities of > 95% neutrophils are typical. Neutrophils are treated with a library of kinase inhibitors, followed by flow cytometry to assess the rate of neutrophil apoptosis. This protocol allows for the high-throughput screening of primary human immune cells to identify compounds with a potential to modify neutrophil function, and could be modified to assess other phenotypes if required.