{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,5]],"date-time":"2026-03-05T02:59:10Z","timestamp":1772679550144,"version":"3.50.1"},"reference-count":40,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,1,23]],"date-time":"2023-01-23T00:00:00Z","timestamp":1674432000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R35GM124911, T32GM065085"],"award-info":[{"award-number":["R35GM124911, T32GM065085"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>Techniques for studying the clearance of bacterial infections are critical for advances in understanding disease states, immune cell effector functions, and novel antimicrobial therapeutics. Intracellular killing of <jats:italic>Staphylococcus aureus<\/jats:italic> by neutrophils can be monitored using a <jats:italic>S. aureus<\/jats:italic> strain stably expressing GFP, a fluorophore that is quenched when exposed to the reactive oxygen species (ROS) present in the phagolysosome. Here, we expand upon this method by developing a bi-fluorescent <jats:italic>S. aureus<\/jats:italic> killing assay for use <jats:italic>in vivo.<\/jats:italic> Conjugating <jats:italic>S. aureus<\/jats:italic> with a stable secondary fluorescent marker enables the separation of infected cell samples into three populations: cells that have not engaged in phagocytosis, cells that have engulfed and killed <jats:italic>S. aureus<\/jats:italic>, and cells that have viable internalized <jats:italic>S. aureus<\/jats:italic>. We identified ATTO647N-NHS Ester as a favorable dye conjugate for generating bi-fluorescent <jats:italic>S. aureus<\/jats:italic> due to its stability over time and invariant signal within the neutrophil phagolysosome. To resolve the <jats:italic>in vivo<\/jats:italic> utility of ATTO647N\/GFP bi-fluorescent <jats:italic>S. aureus<\/jats:italic>, we evaluated neutrophil function in a murine model of chronic granulomatous disease (CGD) known to have impaired clearance of <jats:italic>S. aureus<\/jats:italic> infection. Analysis of bronchoalveolar lavage (BAL) from animals subjected to pulmonary infection with bi-fluorescent <jats:italic>S. aureus<\/jats:italic> demonstrated differences in neutrophil antimicrobial function consistent with the established phenotype of CGD.<\/jats:p>","DOI":"10.3389\/fimmu.2023.1089111","type":"journal-article","created":{"date-parts":[[2023,1,23]],"date-time":"2023-01-23T07:10:15Z","timestamp":1674457815000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Bi-fluorescent Staphylococcus aureus infection enables single-cell analysis of intracellular killing in vivo"],"prefix":"10.3389","volume":"14","author":[{"given":"Kristina D.","family":"Hinman","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sonia S.","family":"Laforce-Nesbitt","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joshua 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