{"id":3840,"date":"2025-02-10T00:11:11","date_gmt":"2025-02-10T05:11:11","guid":{"rendered":"https:\/\/www.med.unc.edu\/flowcytometry\/?page_id=3840"},"modified":"2026-04-16T14:47:28","modified_gmt":"2026-04-16T18:47:28","slug":"rigor","status":"publish","type":"page","link":"https:\/\/www.med.unc.edu\/flowcytometry\/rigor\/","title":{"rendered":"Rigor and Reproducibility"},"content":{"rendered":"<div class=\"row  oscitas-bootstrap-container\">\n<div class=\"col-lg-4 col-md-12 col-xs-12 col-sm-12 oscitas-bootstrap-container\">\n<a class=\"btn btn-primary btn-md btn-block  oscitas-bootstrap-container\" target=\"_self\" href=\"#nih\">NIH Rigor &amp; Reproducibility<\/a>\n<\/div>\n<div class=\"col-lg-4 col-md-12 col-xs-12 col-sm-12 oscitas-bootstrap-container\">\n<a class=\"btn btn-primary btn-md btn-block  oscitas-bootstrap-container\" target=\"_self\" href=\"#rigor\">UNC Rigor &amp; Reproducibility<\/a>\n<\/div>\n<div class=\"col-lg-4 col-md-12 col-xs-12 col-sm-12 oscitas-bootstrap-container\">\n<a class=\"btn btn-primary btn-md btn-block  oscitas-bootstrap-container\" target=\"_self\" href=\"#valid\">Antibody Validation<\/a>\n<\/div>\n<\/div>\n<h2 id=\"nih\">Rigor and Reproducibility Resources<\/h2>\n<p>Learn about the <a href=\"http:\/\/grants.nih.gov\/reproducibility\/index.htm\" rel=\"noopener\">NIH Initiative to Enhance Reproducibility through Rigor and Transparency<\/a>. (<a href=\"http:\/\/grants.nih.gov\/grants\/policy\/rigor\/NIH_Policy_Rigor_For_Reviewers\/presentation.html\" rel=\"noopener\">NIH Video<\/a>)<\/p>\n<p><a href=\"https:\/\/grants.nih.gov\/faqs#\/rigor-and-reproducibility.htm\">NIH Rigor and Reproducibility FAQs<\/a><\/p>\n<p class=\"entry-title\">Grant Resource Authentication Plan Resources:<\/p>\n<ul>\n<li>Check out instructions on <a href=\"https:\/\/nexus.od.nih.gov\/all\/2016\/10\/26\/what-kind-of-information-resource-authentication-plan\/?utm_source=nexus&amp;utm_medium=email&amp;utm_content=nihupdate&amp;utm_campaign=oct16\">NIH Nexus Blog<\/a>.<\/li>\n<li><a href=\"https:\/\/nexus.od.nih.gov\/all\/2017\/06\/06\/what-are-key-biological-andor-chemical-resources-that-should-be-addressed-in-my-applications-authentication-plan\/?utm_source=nexus&amp;utm_medium=email&amp;utm_content=nihupdate&amp;utm_campaign=may17\">Key Biological and\/or Chemical Resources<\/a><\/li>\n<\/ul>\n<p><a title=\"\" href=\"https:\/\/www.faseb.org\/FASEB\/media\/PDF\/News\/Washington%20Update\/FASEB_Enhancing-Research-Reproducibility_1.pdf\" target=\"_self\">FASEB report <\/a>on enhancing research reproducibility identifies three main gaps to research reproducibility:<\/p>\n<ul>\n<li>Lack of uniform definitions to describe the problem<\/li>\n<li>Insufficient reporting of key experimental details<\/li>\n<li>Gaps in scientific training<\/li>\n<\/ul>\n<hr \/>\n<h3 id=\"rigor\">Guide to Rigor, Reproducibility, and Transparency for Flow Cytometry Experiments in the UNC Flow Cytometry Core Facility<\/h3>\n<p><span class=\"s1\"><b>A.\u00a0 Eight steps to Rigorous and Reproducible Experiments in Biomolecular Research:<\/b><\/span><\/p>\n<ol class=\"ol2\">\n<li class=\"li2\"><span class=\"s1\">If using a core facility,\u00a0<b>consult\u00a0<\/b>with the core staff in the planning stage. Consult with a statistician if you need help developing a Power Analysis to assure that your results will be adequately powered.<\/span><\/li>\n<li class=\"li2\"><span class=\"s1\">Design your experiment with\u00a0<b>sufficient controls<\/b>\u00a0(rigor)\u00a0<b>and replicates <\/b>(reproducibility).<\/span><\/li>\n<li class=\"li2\"><span class=\"s1\">Assure that ALL of your reagents (antibodies, cell lines, mice) are<b>\u00a0fully validated <\/b>(see below).<\/span><\/li>\n<li class=\"li2\"><span class=\"s1\">Have a clear and\u00a0<b>detailed protoco<\/b>l (SOP) and data analysis plan. Assure that the protocol is strictly followed or that any deviation is well documented.<\/span><\/li>\n<li class=\"li2\"><span class=\"s1\">Assure that the staff or students performing the experiment are\u00a0<b>well trained<\/b>\u00a0and understand each step and the importance of performing them precisely.<\/span><\/li>\n<li class=\"li2\"><span class=\"s1\">Use only\u00a0<b>well-maintained instrumentation<\/b>, preferably maintained and operated in a core facility with expert staff (see #1 above).<\/span><\/li>\n<li class=\"li2\"><span class=\"s1\"><b>Document all steps<\/b>, reagents, equipment and data analysis methods used in the experiment. Assure that the both the documentation and the data itself are properly stored in a safe data management repository.<\/span><\/li>\n<li class=\"li2\"><span class=\"s1\"><b>Acknowledge<\/b> all grants that support the core, the core (by name), and core staff in publications. (See <a href=\"https:\/\/www.med.unc.edu\/flowcytometry\/resources\/publication-acknowledgements\/\">Publication Acknowledgments<\/a>)<\/span><\/li>\n<\/ol>\n<p><b><i><\/i><\/b><span class=\"s1\"><b>B.\u00a0 Guide to Rigor and Reproducibility for the UNC Flow Cytometry Core<\/b><\/span><\/p>\n<ol class=\"ol2\">\n<li class=\"li2\"><span class=\"s1\"><strong>Consult<\/strong> with the core staff in the planning stage. <a href=\"https:\/\/uncch.ilab.agilent.com\/sc\/3267\/unc-flow-cytometry-core-facility\/?tab=services\">Consultations<\/a> are free and can be scheduled through iLabs.<\/span><\/li>\n<li class=\"li2\"><strong>Plan for Rigor<\/strong>: Multicolor Flow Cytometry experiments should be run using replicates [be mindful of biological \/ technical replicates] based on <a title=\"\" href=\"http:\/\/www.powerandsamplesize.com\/\" target=\"_self\">power calculations<\/a> and include appropriate controls.\n<div>\n<ul>\n<li>Include unstained controls and single-color controls with each experiment for sensitivity and compensation calculations. Fluorophore compensation and analysis can be completed on the cytometers or using FlowJo, FCS Express, or similar 3rd party analysis software. Removal of debris, aggregates, and where possible non-relevant cell lineages from single-color control data files may improve the compensation calculation.<\/li>\n<li>Include a <a href=\"https:\/\/www.linkedin.com\/pulse\/3-dead-cell-reagents-improve-your-data-analysis-tim-bushnell\/\">viability dye<\/a> to exclude dead cells from the analysis<\/li>\n<li>Incorporate Biological controls and Fluorescence Minus One (FMO) controls to validate the expression of rare or low-expressing markers.<\/li>\n<li>Run doublet discrimination in your analysis to exclude aggregates<\/li>\n<li>Run time as a parameter to assure that the fluidics were running smoothly during acquisition<\/li>\n<\/ul>\n<\/div>\n<\/li>\n<li class=\"li2\"><strong>Validate Reagents<\/strong>, in particular, all antibodies should be validated prior to reporting of results.<a title=\"\" href=\"https:\/\/www.faseb.org\/FASEB\/media\/PDF\/News\/Washington%20Update\/FASEB_Enhancing-Research-Reproducibility_1.pdf\" target=\"_self\"> Recommendations for research using antibodies<\/a> (page 7-8) &#8216;Although vendor-supplied technical information may help investigators select reagents such as antibodies, this information is insufficient for validation&#8217;. See the section below to assure the use of <a href=\"https:\/\/www.med.unc.edu\/flowcytometry\/resources\/rigor\/#valid\">validated antibodies<\/a>.<\/li>\n<li><strong>Optimize your sample preparation and staining protocols<\/strong>. For help you can reach out to the core for a <a href=\"https:\/\/uncch.ilab.agilent.com\/sc\/3267\/unc-flow-cytometry-core-facility\/?tab=services\">consultation<\/a>, we also provide some starting points on our <a href=\"https:\/\/www.med.unc.edu\/flowcytometry\/resources\/protocols\/\">Flow Cytometry Associated Protocols<\/a> page.\u00a0<b><\/b><\/li>\n<li><a href=\"https:\/\/www.med.unc.edu\/flowcytometry\/services\/training\/\"><strong>Get trained<\/strong><\/a> on the proper use of core instrumentation.\u00a0<b><\/b><\/li>\n<li><strong>Confirm instruments are fit for service<\/strong>. Daily quality control of all instrumentation is performed by staff based on manufacturers\u2019 recommendations to ascertain that all of the UNC Flow Cytometry Core Facility flow cytometers maintain peak performance. In addition to quality control, drop deflection voltages are set, and a drop delay calculation is carried out on all cell sorters prior to sorting.\n<ul class=\"ol2\">\n<li>For both sorting and analysis the core recommend Single-color controls are included when appropriate for compensation calculations.<\/li>\n<li>For sorting the core recommends that Post-sort analyses are run on each population to verify purity when possible.<b><\/b><\/li>\n<\/ul>\n<\/li>\n<li><strong>Properly document and store your flow data<\/strong>. Guidelines for reporting have been adopted by the International Society for Advancement of Cytometry (ISAC). For details see <a href=\"http:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/cyto.a.20623\/pdf\" target=\"_blank\" rel=\"noopener noreferrer\">MIFlowCyt<\/a>\u00a0\u2013 <i>\u2018Minimum Information for Flow Cytometry experiments\u2019\u00a0<\/i>for recommendations on reporting Flow Cytometry Data. Additionally, assure that your data is properly stored in a safe data management repository e.g. <a href=\"http:\/\/flowrepository.org\/\">Flow Repository<\/a> or <a href=\"https:\/\/dataverse.unc.edu\/\">UNC Dataverse<\/a>.\u00a0<b><\/b><\/li>\n<li><span class=\"s1\"><b>Acknowledge<\/b> all grants that support the core, the core (by name), and core staff in publications. (See <a href=\"https:\/\/www.med.unc.edu\/flowcytometry\/resources\/publication-acknowledgements\/\">Publication Acknowledgments<\/a>)<\/span><b><\/b><\/li>\n<\/ol>\n<div>\n<hr \/>\n<\/div>\n<div>\n<h3 id=\"valid\">Antibody Validation<\/h3>\n<p><strong>Antibody Registry\u00a0 \u00a0<\/strong><\/p>\n<p>Check for validated antibodies using <a href=\"https:\/\/www.citeab.com\/\">CiteAb<\/a>, <a href=\"https:\/\/landing.benchsci.com\/academic\">BenchSci<\/a>, or the <a href=\"http:\/\/antibodyregistry.org\/\">Antibody Registry<\/a>, which include commercial antibodies from hundreds of commercial vendors and thousands of individual labs.<\/p>\n<p><strong>Tips for working with Antibodies:<\/strong><\/p>\n<ol>\n<li>Always Titrate<\/li>\n<li>Validate Specificity<\/li>\n<li>Integrate critical controls like Biological controls<\/li>\n<li>See: Uhlen et al., <a title=\"\" href=\"https:\/\/www.nature.com\/articles\/nmeth.3995\" target=\"_self\">A Proposal for Validation of Antibodies<\/a>, Nature Methods (2016)<\/li>\n<li><a title=\"\" href=\"https:\/\/expertcytometry.com\/4-steps-to-validate-flow-cytometry-antibodies-and-improve-reproducibility\/\" target=\"_self\">Consider the Tips provided by Expert Cytometry<\/a><\/li>\n<li>Additional resources are available on the EuroMab network:<\/li>\n<\/ol>\n<p>EuroMAbNet Guidelines and Resources:<\/p>\n<ul>\n<li><a href=\"https:\/\/www.euromabnet.com\/guidelines\/index.php\">EuroMAbNet Guidelines<\/a>; PMID:<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pubmed\/26418356\">26418356<\/a><\/li>\n<li><a href=\"https:\/\/www.euromabnet.com\/guidelines\/positive-negative-controls.php\">Pos\/Neg Controls<\/a><\/li>\n<li><a href=\"https:\/\/www.euromabnet.com\/guidelines\/articles-about-antibody-validation.php\">Additional Articles about Ab Validation<\/a><\/li>\n<\/ul>\n<p>Also, <a title=\"\" href=\"https:\/\/fluorofinder.com\/\" target=\"_self\">FluoroFinder <\/a>has partnered with CiteAb to provide validation data for &gt;200,000 reagents.<\/p>\n<p>Explore OMIPs; Optimized Multicolor Immunofluorescence Panels (OMIPs) are peer-reviewed panels designed for fluorescent assays. Available via <a title=\"\" href=\"https:\/\/fluorofinder.com\/omips\/\" target=\"_self\">Fluorofinder<\/a>, or <a href=\"https:\/\/public.tableau.com\/app\/profile\/fanny2212\/viz\/OMIP_ISAC\/Menu\">Tableau of OMIPs<\/a>. The <a href=\"https:\/\/cancer.wisc.edu\/research\/resources\/flow\/tech-notes-resources\/\">UWCCC Flow Lab<\/a> also maintains an <a href=\"https:\/\/docs.google.com\/spreadsheets\/d\/1PCRSXV_J0CAxKfkRUFwPjp6uq2mdJntCyN-cD0hhI-A\/edit?usp=sharing\">excel sheet of the antibodies used in OMIPs<\/a>.<\/p>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Rigor and Reproducibility Resources Learn about the NIH Initiative to Enhance Reproducibility through Rigor and Transparency. (NIH Video) NIH Rigor and Reproducibility FAQs Grant Resource Authentication Plan Resources: Check out instructions on NIH Nexus Blog. Key Biological and\/or Chemical Resources FASEB report on enhancing research reproducibility identifies three main gaps to research reproducibility: Lack of &hellip; <a href=\"https:\/\/www.med.unc.edu\/flowcytometry\/rigor\/\" aria-label=\"Read more about Rigor and Reproducibility\">Read more<\/a><\/p>\n","protected":false},"author":6662,"featured_media":0,"parent":0,"menu_order":4,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"layout":"","cellInformation":"","apiCallInformation":"","footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-3840","page","type-page","status-publish","hentry","odd"],"acf":[],"_links_to":[],"_links_to_target":[],"_links":{"self":[{"href":"https:\/\/www.med.unc.edu\/flowcytometry\/wp-json\/wp\/v2\/pages\/3840","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.med.unc.edu\/flowcytometry\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.med.unc.edu\/flowcytometry\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/flowcytometry\/wp-json\/wp\/v2\/users\/6662"}],"replies":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/flowcytometry\/wp-json\/wp\/v2\/comments?post=3840"}],"version-history":[{"count":17,"href":"https:\/\/www.med.unc.edu\/flowcytometry\/wp-json\/wp\/v2\/pages\/3840\/revisions"}],"predecessor-version":[{"id":4586,"href":"https:\/\/www.med.unc.edu\/flowcytometry\/wp-json\/wp\/v2\/pages\/3840\/revisions\/4586"}],"wp:attachment":[{"href":"https:\/\/www.med.unc.edu\/flowcytometry\/wp-json\/wp\/v2\/media?parent=3840"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}