Protocol for Evaluating In Vivo the Activation of the P2RX7 Immunomodulator

Biological Procedures Online

Methodology

Thierry Juhel¹  Valérie Vouret-Craviari²  Serena Janho dit Hreich²  Paul Hofman³ 

[1] Université Côte d’Azur, CNRS, INSERM, IRCAN, 28 avenue de Valombrose, 06108, Nice, France;Université Côte d’Azur, CNRS, INSERM, IRCAN, 28 avenue de Valombrose, 06108, Nice, France;FHU OncoAge, Pasteur Hospital, 30 voie Romaine, 06001, Nice, France;Université Côte d’Azur, CNRS, INSERM, IRCAN, 28 avenue de Valombrose, 06108, Nice, France;FHU OncoAge, Pasteur Hospital, 30 voie Romaine, 06001, Nice, France;Laboratory of Clinical and Experimental Pathology and Biobank, Pasteur Hospital, 30 voie Romaine, 06001, Nice, France;Hospital-Related Biobank (BB-0033-00025), Pasteur Hospital, 30 voie Romaine, 06001, Nice, France;

关键词: P2RX7;    P2X7 receptor;    Purinergic receptors;    ATP;    Macropore;    TOPRO-3;    Activity;   

DOI  :  10.1186/s12575-022-00188-6

 received in 2022-08-30, accepted in 2022-12-13,  发布年份 2022

来源: Springer

【 摘 要 】

BackgroundP2RX7 is a purinergic receptor with pleiotropic activities that is activated by high levels of extracellular ATP that are found in inflamed tissues. P2RX7 has immunomodulatory and anti-tumor proprieties and is therefore a therapeutic target for various diseases. Several compounds are developed to either inhibit or enhance its activation. However, studying their effect on P2RX7’s activities is limited to in vitro and ex vivo studies that require the use of unphysiological media that could affect its activation. Up to now, the only way to assess the activity of P2RX7 modulators on the receptor in vivo was in an indirect manner.ResultsWe successfully developed a protocol allowing the detection of P2RX7 activation in vivo in lungs of mice, by taking advantage of its unique macropore formation ability. The protocol is based on intranasal delivery of TO-PRO™-3, a non-permeant DNA intercalating dye, and fluorescence measurement by flow cytometry. We show that ATP enhances TO-PRO™-3 fluorescence mainly in lung immune cells of mice in a P2RX7-dependant manner.ConclusionsThe described approach has allowed the successful analysis of P2RX7 activity directly in the lungs of WT and transgenic C57BL6 mice. The provided detailed guidelines and recommendations will support the use of this protocol to study the potency of pharmacologic or biologic compounds targeting P2RX7.

【 授权许可】

CC BY   
© The Author(s) 2023

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