Herman, K.D., Rahman, A. and Prince, L.R. (2020) Isolation and high throughput flow cytometric apoptosis assay of human neutrophils to enable compound library screening. Bio-protocol, 10 (11). e3640.
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2020 The Authors. This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). |
Keywords: | Apoptosis; Compound library; Flow cytometry; Kinase; Neutrophil; Screen |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Sheffield Teaching Hospitals |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 22 Mar 2021 09:17 |
Last Modified: | 22 Mar 2021 09:48 |
Status: | Published |
Publisher: | Bio-Protocol |
Refereed: | Yes |
Identification Number: | 10.21769/bioprotoc.3640 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:171999 |