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Because of its restricted expression in sperm and crucial role in fertilization, CatSper represents a promising target for the development of nonhormonal male contraceptives. However, its structural complexity and the absence of functional heterologous expression systems have limited inhibitor discovery.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Objectives<\/jats:title>\n                    <jats:p>To develop a scalable and multiplexed flow cytometry assay to identify compounds capable of blocking CatSper activity in mammalian sperm.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Materials and Methods<\/jats:title>\n                    <jats:p>\n                      The assay relies on the stimulation of CatSper by exposing live mouse sperm to a high\u2010K\n                      <jats:sup>+<\/jats:sup>\n                      \/high\u2010pH solution (K8.6), which induces membrane depolarization and intracellular alkalinization, triggering Ca\n                      <jats:sup>2+<\/jats:sup>\n                      entry through CatSper. Changes in intracellular Ca\n                      <jats:sup>2+<\/jats:sup>\n                      levels were monitored using the fluorescent indicator Fluo\u20104. To increase throughput and minimize variability, this assay was combined with fluorescent cell barcoding, which assigns each sperm sample a unique fluorescent signature, enabling the simultaneous acquisition of multiple conditions within a single tube. Additionally, a compound pooling strategy was implemented to further improve assay efficiency, allowing the rapid identification of active compounds within a library. The assay was validated using CatSper1 knockout sperm and known pharmacological inhibitors.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Fluorescent cell barcoding greatly reduced acquisition time and reagent consumption by allowing multiple experimental conditions to be pooled and analyzed simultaneously within a single acquisition. The compound pooling strategy facilitated large\u2010scale screening experiments. Validation confirmed that both genetic deletion and pharmacological blockade of CatSper abolish the K8.6\u2010induced Ca\n                      <jats:sup>2+<\/jats:sup>\n                      response.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>These results establish a robust, scalable, and high\u2010throughput screening platform for identifying novel CatSper blockers and potential male contraceptive candidates.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1111\/andr.70294","type":"journal-article","created":{"date-parts":[[2026,6,22]],"date-time":"2026-06-22T06:40:04Z","timestamp":1782110404000},"page":"1479-1489","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Scalable Flow Cytometry Assay to Evaluate CatSper\u2010mediated Ca\n                    <sup>2+<\/sup>\n                    Influx Triggered by High\u2010K\n                    <sup>+<\/sup>\n                    \/High\u2010pH Stimulation in Mouse Sperm"],"prefix":"10.1111","volume":"14","author":[{"given":"Lucila R.","family":"Gomez\u2010Olivieri","sequence":"first","affiliation":[{"name":"Instituto de Biolog\u00eda y Medicina Experimental (IBYME\u2010CONICET)  Buenos Aires Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liza J.","family":"Schiavi\u2010Ehrenhaus","sequence":"additional","affiliation":[{"name":"Instituto de Biolog\u00eda y Medicina Experimental (IBYME\u2010CONICET)  Buenos Aires Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martina","family":"Jablo\u00f1ski","sequence":"additional","affiliation":[{"name":"Instituto de Biolog\u00eda y Medicina Experimental (IBYME\u2010CONICET)  Buenos Aires Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariano G.","family":"Buffone","sequence":"additional","affiliation":[{"name":"Instituto de Biolog\u00eda y Medicina Experimental (IBYME\u2010CONICET)  Buenos Aires Argentina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guillermina M.","family":"Luque","sequence":"additional","affiliation":[{"name":"Instituto de Biolog\u00eda y Medicina Experimental (IBYME\u2010CONICET)  Buenos Aires 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