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As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within<jats:italic>Leishmania<\/jats:italic>parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5\u2010diaminofluorescein diacetate (DAF\u20102DA)]. Initially, assay optimization was performed in<jats:italic>Leishmania donovani<\/jats:italic>promastigotes, assay specificity being confirmed using both a NO donor [S\u2010nitroso\u2010<jats:italic>N<\/jats:italic>\u2010acetyl\u2010penicillamine (SNAP)] and a NO scavenger [2\u2010(4\u2010carboxyphenyl)\u20104,4,5,5\u2010tetramethylimidazoline\u20101\u2010oxyl\u20103\u2010oxide, C\u2010PTIO]. Using 40 \u03bcM DAF\u20102DA, basal levels of intracellular NO were measured which varied in different<jats:italic>Leishmania<\/jats:italic>species; addition of conventional anti\u2010leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF\u20102T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF\u20102DA, being 2 \u03bcM. Following parasitization, levels of NO decreased which was normalized following treatment with anti\u2010leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti\u2010leishmanial compounds. \u00a9 2010 International Society for Advancement of Cytometry<\/jats:p>","DOI":"10.1002\/cyto.a.21001","type":"journal-article","created":{"date-parts":[[2010,12,14]],"date-time":"2010-12-14T14:29:42Z","timestamp":1292336982000},"page":"35-45","source":"Crossref","is-referenced-by-count":47,"title":["Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis"],"prefix":"10.1002","volume":"79A","author":[{"given":"Avijit","family":"Sarkar","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Piu","family":"Saha","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Goutam","family":"Mandal","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Debanjan","family":"Mukhopadhyay","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Susmita","family":"Roy","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sunny Kumar","family":"Singh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sukhen","family":"Das","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"R. 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