Abstract
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.
Highlights
Neutrophils are isolated from human blood by dextran sedimentation and discontinuous plasma/Percoll gradient centrifugation, based on previous protocols (Haslett et al, 1985)
Neutrophils are extremely sensitive to activation, with respect to rates of cell death, so minor modifications have been made in order to minimize handling and activation of cells
We have developed a flow cytometric assay using the Attune Autosampler to allow us to screen compound libraries in a fast and efficient way, using Annexin-V and TOPRO-3 as markers of apoptosis
Summary
90 mm diameter x 15 mm deep (Thermo Fisher Scientific, catalog number: 101R20) 2. 15 ml polypropylene tubes (SARSTEDT, catalog number: 62.554.502) 3. 50 ml polypropylene tubes (SARSTEDT, catalog number: 62.547.254) 4. 1.5 ml centrifuge tubes 5. 0.2 μm syringe filter 6. 90 mm diameter x 15 mm deep (Thermo Fisher Scientific, catalog number: 101R20) 2. BD Plastipak Eccentric Luer-Slip Syringe (Fisher Scientific, catalog number: 300866) 10. Serological pipettes (Fisher Scientific, catalog number: 13-676-10J) 11. Hank’s Balanced Salt Solution (Gibco, Life Technologies, catalog number: 14170-112) 17. Penicillin-streptomycin (Gibco, Life Technologies, catalog number: 15140122) 19. 1x Phosphate Buffered Saline (Gibco, Life Technologies, catalog number: 10010023) 20. Annexin binding buffer (ABB) (BD Pharmingen, BD Biosciences, catalog number: 556454) 23. TOPRO-3 (Molecular Probes, Life Technologies, catalog number: T3605) 26. Ethanol absolute (Fisher Scientific, catalog number: 10428671) 29. Kwik-Diff Solution 3 (Thermo Fisher Scientific, catalog number: 9990707) 31. Heraeus Biofuge Fresco (Thermo Fisher Scientific, catalog number: 75005521) 6. Cytospin 4 cytocentrifuge (Thermo Fisher Scientific, catalog number: A78300003).
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have