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The protocol describes systematic isolation of leaflet-derived cells from pulmonary and aortic valves, enzymatic digestion to obtain single-cell suspensions, and preparation for antibody and viability staining. It outlines gating strategies to enable accurate, reproducible quantification of infiltrating immune cells. The workflow addresses technical constraints of valve tissue and GFP-associated background fluorescence to preserve interpretation of staining results.",{"@graph":69,"@context":122},[70,84,105],{"@type":71,"itemListElement":72},"BreadcrumbList",[73,77,79,82],{"item":74,"name":75,"@type":76,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":78,"name":9,"@type":76,"position":14},"https://docshare.wps.com/document/",{"item":80,"name":35,"@type":76,"position":81},"https://docshare.wps.com/document/healthcare/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/a-step-by-step-protocol-for-isolation-and-flow-cytometry-analysis-of-porcine-cardiac-valve-leaflets/411803/",{"url":83,"name":65,"@type":85,"image":86,"author":91,"headline":65,"publisher":94,"fileFormat":97,"inLanguage":63,"description":67,"dateModified":98,"datePublished":99,"encodingFormat":97,"isAccessibleForFree":100,"interactionStatistic":101},"DigitalDocument",{"url":87,"@type":88,"width":89,"height":90},"https://docshare.wps.com/thumbnails/a-step-by-step-protocol-for-isolation-and-flow-cytometry-analysis-of-porcine-cardiac-valve-leaflets/411803.png","ImageObject",300,407,{"name":92,"@type":93},"Aran","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-11-02","2026-09-28",true,{"@type":102,"interactionType":103,"userInteractionCount":44},"InteractionCounter",{"@type":104},"ViewAction",{"@type":106,"mainEntity":107},"FAQPage",[108,114,118],{"name":109,"@type":110,"acceptedAnswer":111},"What is the main purpose of this flow cytometry protocol for porcine valve leaflets?","Question",{"text":112,"@type":113},"To enable systematic isolation and phenotypic characterization of leaflet-derived cells and to support accurate, reproducible flow cytometric analysis in GFP-expressing porcine valve models used for partial heart transplantation immunobiology.","Answer",{"name":115,"@type":110,"acceptedAnswer":116},"Which steps are included to prepare samples for flow cytometry?",{"text":117,"@type":113},"The protocol covers valve procurement and transportation, enzymatic digestion to generate a single-cell suspension, antibody and viability staining, and subsequent sample acquisition for analysis.",{"name":119,"@type":110,"acceptedAnswer":120},"Why does the protocol emphasize GFP-specific considerations and gating strategies?",{"text":121,"@type":113},"Because ubiquitously expressed GFP alters background fluorescence, potentially overlapping with viability and marker signals; GFP-tailored interpretation and gating strategies are used to ensure reliable assessment of staining results.","https://schema.org",{"og:url":83,"og:type":124,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":126,"canonical":83},"index,follow",{"doc_id":128,"site_id":62},411803,1790742817,{"code":4,"msg":5,"data":131},{"doc_id":128,"user_id":132,"nickname":92,"user_avatar":133,"doc_module":4,"category_id":34,"category_name":35,"doc_title":65,"doc_description":67,"doc_content":134,"file_id":135,"file_url":136,"file_type":137,"file_size":138,"view_count":44,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":56,"language":139,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":140,"faqs":141,"seo_title":142,"seo_description":67,"update_tm":143,"read_time":144,"preview_page_count":4},137455076865,"https://ap-avatar.wpscdn.com/davatar_29158cc5080c5b710cf443261637dec0","ll  \nOPEN ACCESS  \nProtocol  \nA step-by-step protocol for isolation and flow cytometry analysis of porcine cardiac valve leaflets  \nValve  \n45 mins 2 hrs  \nValve procurement & transportation  \nAntibody & viability staining  \nStep 1 Step 2 Step 3 Step 4  \nTissue processing & digestion  \nSample acquisition & analysis  \n3 hrs 20 mins  \nCollagenase P  \nFlow Cytometer  \n70µm Filter  \nSingle cells  \nGFP-expressing porcine valves are indispensable models for partial heart transplantation. Here, we present a protocol for the systematic isolation and phenotypic characterization of leafletderived cells from pulmonary and aortic valves. We describe steps for valve procurement, enzymatic digestion to single-cell suspension, preparation of samples with antibody, and viability staining and gating strategies for accurate and reproducible flow cytometric analysis. This approach provides valuable insight into valve biology and supports rigorous evaluation of cellular dynamics in experimental transplantation models.  \nPublisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.  \nHerra Javed, Zachary Waldrip, Anshaal Furrukh,   , Kristin Whitworth, Marie Burdine, T. Konrad Rajab  \n[hjaved@uams.edu](hjaved@uams.edu) (H.J.)  \n[tkrajab@uams.edu](tkrajab@uams.edu) (T. K. R.)  \nHighlights  \nSteps for digestion of heart valve leaflets to single-cell suspension  \nDirections for preparing sample with antibody and viability staining with controls  \nGuidance on gating strategies to assess cellular viability and immune response  \nInstructions for statistical analysis  \nJaved et al. , STAR Protocols 6 , 104242  \nDecember 19, 2025 © 2025 The Author(s) . Published by Elsevier Inc.  \n[https://doi.org/10.1016/](https://doi.org/10.1016/)[ ](https://doi.org/10.1016/)[j.xpro.2025.104242](j.xpro.2025.104242)  \nll  \nOPEN ACCESS  \nProtocol  \nA step-by-step protocol for isolation and flow cytometry analysis of porcine cardiac valve leaflets  \nHerra Javed,1,3,5,* Zachary Waldrip,2,3 Anshaal Furrukh, 1 Eli Contorno, 1 Louis Steen, 1 John Lowery,3 David Irby,3 Randall Rainwater,2,3 Kristin Whitworth,4 Marie Burdine,2,3 and T. Konrad Rajab 1,6,*  \n1Department of Cardiovascular Surgery, Arkansas Children’s Hospital, Little Rock, AR 72202, USA  \n2Division of Surgical Research, University of Arkansas for Medical Sciences, Little Rock, AR 72202, USA  \n3Arkansas Children’s Research Institute, Little Rock, AR 72202, USA  \n4National Swine Resource and Research Center, University of Missouri, Columbia, MO 65211, USA  \n5Technical contact  \n6Lead contact  \n*Correspondence: [hjaved@uams.edu](hjaved@uams.edu) (H.J.), [tkrajab@uams.edu](tkrajab@uams.edu) (T. K. R.) [https://doi.org/10.1016/j.xpro.2025.104242](https://doi.org/10.1016/j.xpro.2025.104242)  \nSUMMARY  \nFlow cytometry analysis of GFP-expressing porcine valves is an indispensable model for studying partial heart transplantation immunobiology. Here, we present a protocol for the systematic isolation and phenotypic characterization of leaflet-derived cells from pulmonary and aortic valves. We describe steps for valve procurement, enzymatic digestion to single-cell suspension, preparation of samples with antibody, and viability staining and gating strategies for accurate and reproducible flow cytometric analysis. This approach provides valuable insight into valve biology and supports rigorous evaluation of cellular dynamics  \nin experimental transplantation models.  \nBEFORE YOU BEGIN  \nDue to the unique similarities that swine share with humans, they have become the favored animal model for cardiovascular research, including research involving the heart valves.1 The establishment of techniques for various isolation and characterization tests means that a range of options can be used to gain data on porcine valves in the research setting.2 ,3 However, no established protocol for flow cytometry of porcine valves currently exists. This protocol was ","cbCaiqt6GIq0KL9l","https://ap.wps.com/l/cbCaiqt6GIq0KL9l","pdf",8844297,"English","# Valve procurement & transportation\n# Tissue processing & digestion\n# Antibody & viability staining\n# Sample acquisition & analysis\n## Gating strategies\n## Single-cell suspension and filter preparation\n# Innovation and workflow validation\n# Laser settings and instrument parameters","[{\"question\":\"What is the main purpose of this flow cytometry protocol for porcine valve leaflets?\",\"answer\":\"To enable systematic isolation and phenotypic characterization of leaflet-derived cells and to support accurate, reproducible flow cytometric analysis in GFP-expressing porcine valve models used for partial heart transplantation immunobiology.\"},{\"question\":\"Which steps are included to prepare samples for flow cytometry?\",\"answer\":\"The protocol covers valve procurement and transportation, enzymatic digestion to generate a single-cell suspension, antibody and viability staining, and subsequent sample acquisition for analysis.\"},{\"question\":\"Why does the protocol emphasize GFP-specific considerations and gating strategies?\",\"answer\":\"Because ubiquitously expressed GFP alters background fluorescence, potentially overlapping with viability and marker signals; GFP-tailored interpretation and gating strategies are used to ensure reliable assessment of staining results.\"}]","A step-by-step protocol for isolation and flow cytometry analysis of porcine cardiac valve leaflets | PDF",1790589645,48]