Real-Time Flow Cytometry as a Tool to Monitor Cellular Consequences of P2X7 Activation in Multiple Cell Populations Mixed in a Single FACS Tube.
ATP
Flow cytometry
Microglia
P2X7
T-cell
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
1
7
2022
pubmed:
2
7
2022
medline:
8
7
2022
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 35776332
doi: 10.1007/978-1-0716-2384-8_16
doi:
Substances chimiques
Inflammasomes
0
Adenosine Triphosphate
8L70Q75FXE
Calcium
SY7Q814VUP
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
291-302Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Références
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