Real-Time Flow Cytometry as a Tool to Monitor Cellular Consequences of P2X7 Activation in Multiple Cell Populations Mixed in a Single FACS Tube
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Real-Time Flow Cytometry as a Tool to Monitor Cellular Consequences of P2X7 Activation in Multiple Cell Populations Mixed in a Single FACS Tube. / Veltkamp, Alexander W; Magnus, Tim; Rissiek, Björn.
The P2X7 Receptor: Methods and Protocols. Hrsg. / Annette Nicke. 1. Aufl. New York, NY : HUMANA PRESS INC, 2022. S. 291-302 (Methods Mol Biol; Band 2510).Publikationen: SCORING: Beitrag in Buch/Sammelwerk › SCORING: Beitrag in Sammelwerk › Forschung › Begutachtung
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TY - CHAP
T1 - Real-Time Flow Cytometry as a Tool to Monitor Cellular Consequences of P2X7 Activation in Multiple Cell Populations Mixed in a Single FACS Tube
AU - Veltkamp, Alexander W
AU - Magnus, Tim
AU - Rissiek, Björn
N1 - © 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2022
Y1 - 2022
N2 - The P2X7 receptor is an ATP-gated ion channel expressed by cells of the immune system. In murine T cells, P2X7 activation by high concentrations of ATP or by covalent ADP-ribosylation are potent triggers of cell death. In innate immune cells, such as macrophages or brain microglia, P2X7 is a key regulator of inflammasome activation and the release of mature interleukin 1 beta. ATP-mediated P2X7 activation is accompanied by several direct downstream events, including the influx of calcium, pore formation at the plasma membrane, ectodomain shedding, and cell shrinkage. With this chapter we provide a protocol to monitor all these immediate consequences of P2X7 activation in a time dependent fashion using real-time flow cytometry. We illustrate, for example, how to simultaneously monitor calcium influx and shedding of CD27 in four T cell subpopulations and how to simultaneously analyze calcium influx, pore formation and cell shrinkage in mouse primary microglia. We further provide an extended protocol to compare consequences of P2X7 activation among identical cell populations from two or more different donor mice mixed in a single FACS tube. Taken together, the here presented real-time flow cytometry protocol for measuring P2X7 activation is flexible, scalable and can easily be transferred to other experimental settings.
AB - The P2X7 receptor is an ATP-gated ion channel expressed by cells of the immune system. In murine T cells, P2X7 activation by high concentrations of ATP or by covalent ADP-ribosylation are potent triggers of cell death. In innate immune cells, such as macrophages or brain microglia, P2X7 is a key regulator of inflammasome activation and the release of mature interleukin 1 beta. ATP-mediated P2X7 activation is accompanied by several direct downstream events, including the influx of calcium, pore formation at the plasma membrane, ectodomain shedding, and cell shrinkage. With this chapter we provide a protocol to monitor all these immediate consequences of P2X7 activation in a time dependent fashion using real-time flow cytometry. We illustrate, for example, how to simultaneously monitor calcium influx and shedding of CD27 in four T cell subpopulations and how to simultaneously analyze calcium influx, pore formation and cell shrinkage in mouse primary microglia. We further provide an extended protocol to compare consequences of P2X7 activation among identical cell populations from two or more different donor mice mixed in a single FACS tube. Taken together, the here presented real-time flow cytometry protocol for measuring P2X7 activation is flexible, scalable and can easily be transferred to other experimental settings.
U2 - 10.1007/978-1-0716-2384-8_16
DO - 10.1007/978-1-0716-2384-8_16
M3 - SCORING: Contribution to collected editions/anthologies
C2 - 35776332
SN - 978-1-0716-2383-1
T3 - Methods Mol Biol
SP - 291
EP - 302
BT - The P2X7 Receptor
A2 - Nicke, Annette
PB - HUMANA PRESS INC
CY - New York, NY
ER -
