High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding
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Le résumé fourni par la source
The exclusive expression of CatSper in sperm and its critical role in sperm function makes this channel an attractive target for contraception. The strategy of blocking CatSper as a male, non-hormonal contraceptive has not been fully explored due to the lack of robust screening methods to discover novel and specific inhibitors. The reason for this lack of appropriate methodology is the structural and functional complexity of this channel. We have developed a high-throughput method to screen drugs with the capacity to block CatSper in mammalian sperm. The assay is based on removing external free divalent cations by chelation, inducing CatSper to efficiently conduct monovalent cations. Since Na+ is highly concentrated in the extracellular milieu, a sudden influx depolarizes the cell. Using CatSper1 KO sperm we demonstrated that this depolarization depends on CatSper function. A membrane potential (Em) assay was combined with fluorescent cell barcoding (FCB), enabling higher throughput flow cytometry based on unique fluorescent signatures of different sperm samples. These differentially labeled samples incubated in distinct experimental conditions can be combined into one tube for simultaneous acquisition. In this way, acquisition times are highly reduced, which is essential to perform larger screening experiments for drug discovery using live cells. Altogether, a simple strategy for assessing CatSper was validated, and this assay was used to develop a high-throughput drug screening for new CatSper blockers.
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Le contrôle bibliographique ouvert
DOI retrouvé dans Crossref DOI retrouvé ; titre concordant.
- Titre Crossref
- High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding
- Date Crossref
- 19/01/2023
- Éditeur
- Frontiers Media SA
- Type
- journal-article
Ce recoupement confirme des métadonnées liées au DOI. Il ne confirme ni la méthode ni les conclusions de l’étude, et il ne compte pas comme une seconde source scientifique indépendante.
Où se fait cette recherche
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Consejo Nacional de Investigaciones Científicas y Técnicas pays non établi dans la noticeOrganisme public
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Experimental Medicine and Biology Institute pays non établi dans la noticeStructure de recherche
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Instituto de Biología Molecular y Celular de Rosario pays non établi dans la noticeStructure de recherche
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National University of General San Martín pays non établi dans la noticeUniversité ou école supérieure
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Instituto de Biología y Medicina Experimental (IBYME-CONICET) pays non établi dans la noticeStructure de recherche
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Instituto de Investigaciones Biotecnológicas pays non établi dans la noticeStructure de recherche
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Instituto de Biotecnología pays non établi dans la noticeStructure de recherche
Consejo Nacional de Investigaciones Científicas y Técnicas, Experimental Medicine and Biology Institute et Instituto de Biología Molecular y Celular de Rosario, avec 4 autres affiliations.
Une affiliation ne permet pas de déduire la nationalité d’un auteur.