Abstract
Multiplexed phosphoflow cytometry is a novel method that provides rapid and quantitative readouts on intracellular phosphoprotein signaling. In this approach, flow cytometry is combined with fluorescent cell barcoding (FCB) to facilitate high-throughput analyses of signaling events. After stimulation, fixed and permeabilized platelets are labeled with distinct dye intensities to create unique fluorescent signatures for individual samples. These uniquely labeled samples can be combined for simultaneous antibody staining and acquisition. During software analysis, multiplexed samples can be differentiated by their distinct fluorescence intensities and analyzed as if they had been acquired individually. Multiplexing eliminates intersample variation, increases statistical robustness, and allows 4–96 samples to be processed with no appreciable increase in antibody consumption or runtime. The method can be performed on washed platelets, platelet-rich plasma (PRP), and whole blood. Its inherent versatility can fulfil wide-ranging experimental requirements from simple dose titrations to complex pharmacologic screens.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Dittrich M, Birschmann I, Mietner S et al (2008) Platelet protein interactions: map, signaling components, and phosphorylation groundstate. Arterioscler Thromb Vasc Biol 28:1326–1331
Spurgeon BEJ, Aburima A, Oberprieler N et al (2014) Multiplexed phosphospecific flow cytometry enables large-scale signaling profiling and drug screening in blood platelets. J Thromb Haemost 12:1733–1743
Krutzik PO, Nolan GP (2006) Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods 3:361–368
Kotecha N, Krutzik PO, Irish JM (2010) Web-based analysis and publication of flow cytometry experiments. Curr Protoc Cytom. https://doi.org/10.1002/0471142956.cy1017s53
Stephen P Perfetto, David Ambrozak, Richard Nguyen, Pratip K Chattopadhyay, Mario Roederer, (2012) Quality assurance for polychromatic flow cytometry using a suite of calibration beads. Nature Protocols 7(12):2067–2079
Krutzik PO, Nolan GP (2003) Intracellular phosphoprotein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A 55:61–70
Davies R, Vogelsang P, Jonsson R et al (2016) An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells. J Immunol Methods 436:58–63
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Spurgeon, B.E.J., Naseem, K.M. (2018). High-Throughput Signaling Profiling in Blood Platelets by Multiplexed Phosphoflow Cytometry. In: Gibbins, J., Mahaut-Smith, M. (eds) Platelets and Megakaryocytes . Methods in Molecular Biology, vol 1812. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8585-2_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8585-2_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8584-5
Online ISBN: 978-1-4939-8585-2
eBook Packages: Springer Protocols