Abstract
Combining density gradient centrifugation with magnetic cell sorting provides a powerful tool to isolate blood cells with high reproducibility, yield, and purity. It also allows for subsequent separation of multiple cell types, resulting in the possibility to analyze different purified fractions from one donor’s sample. The centrifugation step divides whole blood into peripheral blood mononuclear cells (PBMC), erythrocytes, and platelet-rich plasma. In the following, lymphocyte subtypes can be consecutively isolated from the PBMC fraction. This chapter describes enrichment of erythrocytes, CD14-positive monocytes and CD3-positive T lymphocytes. Alternatively, other cell types can be targeted by using magnetic beads specific for the desired subpopulation.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Blonder J, Issaq HJ, Veenstra TD (2011) Proteomic biomarker discovery: it’s more than just mass spectrometry. Electrophoresis 32:1541–1548
Mallick P, Kuster B (2010) Proteomics: a pragmatic perspective. Nat Biotechnol 28:695–709
Bennett S, Breit SN (1994) Variables in the isolation and culture of human monocytes that are of particular relevance to studies of HIV. J Leukoc Biol 56:236–240
Dainiak MB, Kumar A, Galaev IY et al (2007) Methods in cell separations. Adv Biochem Eng Biotechnol 106:1–18
Syrovets T, Tippler B, Rieks M et al (1997) Plasmin is a potent and specific chemoattractant for human peripheral monocytes acting via a cyclic guanosine monophosphate-dependent pathway. Blood 89:4574–4583
de Almeida MC, Silva AC, Barral A et al (2000) A simple method for human peripheral blood monocyte isolation. Mem Inst Oswaldo Cruz 95:221–223
Gorelik L, Kauth M, Gehlhar K et al (2008) Modulation of dendritic cell function by cowshed dust extract. Innate Immun 14:345–355
Mayer A, Lee S, Lendlein A et al (2011) Efficacy of CD14(+) blood monocytes/macrophages isolation: positive versus negative MACS protocol. Clin Hemorheol Microcirc 48:57–63
Brosseron F, May C, Schoenebeck B et al (2012) Stepwise isolation of human peripheral erythrocytes, T lymphocytes, and monocytes for blood cell proteomics. Proteomics Clin Appl 6:497–501
Acknowledgement
This work was supported by P.U.R.E. (Protein Unit for Research in Europe), a project of Nordrhein-Westfalen, a federal state of Germany, the Bundesministerium für Bildung und Forschung (NGFNplus, FZ 01GS08143), Germany, and the German Center for Neurodegenerative Diseases (DZNE e.V.) within the Helmholtz Association.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Brosseron, F., Marcus, K., May, C. (2015). Isolating Peripheral Lymphocytes by Density Gradient Centrifugation and Magnetic Cell Sorting. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 1295. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2550-6_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2550-6_3
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2549-0
Online ISBN: 978-1-4939-2550-6
eBook Packages: Springer Protocols