multiple flow cytometry dot plots of different cell populations in immunophenotyping experiment

This section provides educational resources and tools for immunophenotyping by flow cytometry. These resources include a panel design tool, review articles, application notes, protocols, fluorophore guides and more.

Immunophenotyping features

Review article

European Journal of Immunology Cover Volume 47 Issue 10

Guidelines for the use of flow cytometry and cell sorting in immunological studies

This special edition of the European Journal of Immunology is a comprehensive resource prepared by flow cytometry and immunology research experts from around the world. The Flow Cytometry Guidelines not only highlight the latest trends and best practices in basic and advanced flow cytometry but also re‐enforce optimal sample preparation, as well as data analysis, enabling the correct use of state of the art flow cytometry for all involved in immunological research

Panel design tool

Screen view of the Thermo Fisher Scientific flow cytometry panel design tool with viability dye and 2 antibody options for the violet and the blue lasers

Flow Cytometry Panel Builderbeta

This online tool guides you through flow cytometry panel design, providing a simplified, customizable experience to fit your flow cytometry panel design needs.


Immunophenotyping subtopics


Immunophenotyping learning resources

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Type Title Categories
Application note (2017)Validated 10-color T cell panel using the Attune NxT Flow Cytometerantibodies, Attune/Attune NxT Flow Cytometer, flow cytometry, immunology, immunophenotyping, fluorescent dyes, multicolor flow cytometry
Application note (2017) Detection of platelets in whole blood using the Attune NxT Flow Cytometer Attune NxT, flow cytometer, platelets, immunophenotyping, no-wash, no-lyse
Application note (2015)Attune NxT Flow Cytometer for 6-color immunophenotyping analysis of stained human whole blood using a no-lyse, no-wash protocol, with no compensationAttune NxT, flow cytometer, immunophenotyping, multicolor flow cytometry
Application note (2015)Detection of murine regulatory T cells on the Attune NxT Flow CytometerAttune NxT, flow cytometer, immunophenotyping, multicolor flow cytometry
Application note (2015)Multiparameter analysis of murine regulatory T cells and dendritic cells with the Attune NxT Flow CytometerAttune NxT, flow cytometer, immunophenotyping, multicolor flow cytometry
Application note (2015)Ten-parameter immunophenotyping of human lysed whole blood with the Attune NxT Flow Cytometer allows identification of multiple T cell subsets and myeloid cellsAttune NxT, flow cytometer, immunophenotyping, multicolor flow cytometry
Application note (2015) 13-parameter immunophenotyping of human lysed whole blood with the Attune NxT Flow Cytometer allows for the identification of B cells, NK cells, multiple T cell subsets and myeloid cells Alexa Fluor, antibodies, Attune NxT, compensation, flow cytometer, fluorescent dyes, immunophenotyping, multicolor flow cytometry, nanocrystals, Qdot
BioProbes article (2018)Immuno-oncology: Advances in Basic Research and Translational Medicine. With a focus on immune checkpoint inhibitors and T cell immunotherapyantibodies, ELISA, flow cytometry, fluorescence microscopy/fluorescence imaging, high content analysis (HCA), immunohistochemistry (IHC), immunology, immuno-oncology, immunophenotyping, Luminex, ProCartaPlex, spheroids
BioProbes article (2018)A comprehensive resource for state-of-the-art flow cytometry methods. Guidelines for the use of flow cytometry and cell sorting in immunological studiesantibodies and proteins, cell counting, cell cycle, cell proliferation, flow cytometer calibration, flow cytometry, flow cytometry compensation, flow cytometry sample preparation, immunology, immunophenotyping, viability
BioProbes article (2017)Harness immune checkpoints to combat tumors—Immune checkpoint antibodies for flow cytometry, IHC, and functional bioassaysantibodies, cancer, flow cytometry, immunofluorescence (IF), immunohistochemistry (IHC)
BioProbes article (2017)Bright fluorescent polymer dyes for the violet laser—Super Bright antibody conjugatesantibodies, antibody labeling, Attune NxT, flow cytometry, fluorescence, immunophenotyping, multicolor flow cytometry, Super Bright
BioProbes article (2017)T lymphocyte immunophenotyping—14-color flow cytometry panel featuring Super Bright antibody conjugatesantibodies, Attune NxT, immunology, immunophenotyping, multicolor flow cytometry, Super Bright
BioProbes article (2016)Transcription factor expression during differentiation of hPSC-derived cardiomyocytes—A multiparametric approach using the Attune NxT Flow CytometerAttune NxT, flow cytometer, fluorescence, large cell analysis, rare event detection, stem cell research
BioProbes article (2016)Optimized multicolor immunofluorescence panels (OMIPs)—Jump-start your experimental design with published antibody and reagent panelsflow cytometry, fluorescence, immunophenotyping, multicolor flow cytometry
BioProbes article (2015)Isolation and characterization of exosomes using magnetic beadsantibodies, exosomes, flow cytometry, fluorescence, immunophenotyping, multiplex ELISA/Luminex,  particles, protein blotting, western detection
BioProbes article (2015)No-compensation immunophenotyping using a four-laser flow cytometerAttune NxT, flow cytometer, fluorescence, immunophenotyping, multicolor flow cytometry
BioProbes article (2014)Multiplex your cell proliferation assay with GFP, RFP, and R-PE probes—Click-iT Plus EdU Proliferation Kits for imaging and flow cytometrycell proliferation, Click-iT, cell proliferation, multicolor flow cytometry
BioProbes article (2013)Checking vital signs: Don't let dead cells mislead you—Molecular Probes cell health assays for flow cytometryapoptosis, caspase substrate, cell cycle, cell proliferation, cell tracking, Click-iT, flow cytometry, fluorescence, immunophenotyping, oxidative stress, viability,
BioProbes article (2010)Dead cell identification in flow cytometryflow cytometry, fluorescence, immunophenotyping, viability
BioProbes article (2010)Optimal pairing of dyes and antibodies for flow cytometryantibodies, fluorescence, multicolor flow cytometry, particles Qdot
BioProbes article (2009) Novel solid-phase antibody labeling Alexa Fluor, antibody labeling, flow cytometry, fluorescent dyes, fluorescence microscopy/fluorescence imaging, sample preparation, Zenon
BioProbes article (2008) Single-step isolation of functional human NK cells antibodies, cell isolation, flow cytometry, fluorescent dyes, immunology, magnetic beads, sample preparation
BioProbes article (2008) Using Qdot nanocrystal primary antibody conjugates in flow cytometry  fluorescent dyes, multicolor flow cytometry, nanocrystals, Qdot, sample preparation, set up
BioProbes article (2007) Quantum dots as replacements for tandem dyes in flow cytometry  fluorescent dyes, multicolor flow cytometry, nanocrystals, protein labeling, Qdot
BioProbes article (2007) Simultaneously visualize surface and intracellular markers antibody labeling, cell fixatives, flow cytometry, fluorescent dyes, sample preparation, Zenon
BioProbes article (2006) A guide to antibody labeling and detection Alexa Fluor, antibody labeling, flow cytometry, fluorescent dyes, fluorescence microscopy/fluorescence imaging, nanocrystals, Qdot, Zenon
BioProbes article (2006) Multicolor immunodetection using the violet laser Alexa Fluor, antibody labeling, flow cytometry, fluorescent dyes, immunophenotyping, multicolor flow cytometry, nanocrystals, Qdot
Blog postSuper Bright conjugates for flow cytometryantibodies, Attune NxT, flow cytometry, immunophenotyping, multicolor flow cytometry, Super Bright
FluorophorePacific Blue dyeantibody conjugates, flow cytometry, immunophenotyping, violet laser
FluorophorePacific Green dyeantibody conjugates, flow cytometry, immunophenotyping, violet laser
FluorophorePacific Orange dyeantibody conjugates, flow cytometry, immunophenotyping, violet laser
FluorophoreSuper Bright 436 dyeantibody conjugates, flow cytometry, immunophenotyping, tandem polymer dyes, violet laser
FluorophoreSuper Bright 600 dyeantibody conjugates, flow cytometry, immunophenotyping, tandem polymer dyes, violet laser
FluorophoreSuper Bright 645 dyeantibody conjugates, flow cytometry, immunophenotyping, tandem polymer dyes, violet laser
FluorophoreSuper Bright 702 dyeantibody conjugates, flow cytometry, immunophenotyping, tandem polymer dyes, violet laser
FluorophoreAllophycocyanin (APC)antibody conjugates, flow cytometry, immunophenotyping, phycobiliproteins, tandem dyes
FluorophoreR-phycoerythrin (R-PE)antibody conjugates, flow cytometry, immunophenotyping, phycobiliproteins, tandem dyes
FluorophoreCyan fluorescent protein (CFP)flow cytometry, fluorescent proteins
FluorophoreGreen fluorescent protein (GFP)flow cytometry, fluorescent proteins
FluorophoreRed fluorescent protein (RFP)flow cytometry, fluorescent proteins
FluorophoreFluorophore selection guideantibody conjugates, flow cytometry, immunophenotyping
Guided learningT cell stimulation and proliferation eLearning courseantibodies, cell proliferation, cell stimulation, flow cytometry, immunoassays, immunology, immunophenotyping, T cells
Guided learningAntibody validation eLearning courseantibodies
Molecular Probes HandbookPhycobiliproteins—Section 6.4antibody labeling, multicolor flow cytometry, tandem conjugates
Molecular Probes HandbookUsing the Fluorescence SpectraViewer—Note 23.1flow cytometry, fluorescence, fluorescence microscopy/fluorescence imaging, fluorescent dyes, fluorescent proteins
Molecular Probes HandbookZenon Technology: Versatile reagents for immunolabeling—Section 7.3antibody labeling, flow cytometry, fluorescence microscopy/fluorescence imaging, secondary detection, Zenon
Immunology informationFoxp3 overviewflow cytometry, Foxp3,intracellular staining
ProtocolsCell preparation for flow cytometry protocols (Invitrogen eBioscience reagents)flow cytometry, sample preparation
ProtocolsRed blood cell lysis protocols using eBioscience lysis buffers (Invitrogen eBioscience reagents)flow cytometry, sample preparation
ProtocolsStaining cell surface targets for flow cytometry (Invitrogen eBioscience reagents)extracellular staining, flow cytometry, immunophenotyping
ProtocolsStaining intracellular antigens for flow cytometry (Invitrogen eBioscience reagents)flow cytometry, immunophenotyping, intracellular staining
Protocols Super Bright staining buffer protocol antibody conjugates, flow cytometry, immunophenotyping
Scientific poster (2018) Modular, defined assembly of antibody-based constructs using click chemistry Alexa Fluor, antibody labeling, Attune/Attune NxT, CellInsight, drug discovery, drug research, high content analysis (HCA), SiteClick, spheroids
Scientific poster (2018) Prospective cytometric study evaluating the alkaline phosphatase activity as a potential functional biomarker of primitive malignant cells in acute leukemia antibodies, Attune/Attune NxT, cancer, cell function, flow cytometry, immunophenotyping, stem cell research
Scientific Poster (2017) T-lymphocyte immunophenotyping: 14-color flow cytometry panel design using the Attune NxT Flow Cytometer and Super Bright fluorescent dyes antibodies, Attune NxT, fluorescent dyes, immunophenotyping, multicolor flow cytometry, particles, Super Bright
Scientific Poster (2015) A no lyse, no-wash approach to characterizing phagocyte phenotype and function in whole human blood on the Attune NxT Flow Cytometer Attune NxT, cell cycle, flow cytometer, flow cytometry assays, immunophenotyping, no lyse, phagocytosis, whole blood
Scientific poster (2014)Copper-safe Click-iT Plus EdU proliferation assay: Improved compatibility with simultaneous phycobiliprotein and fluorescent protein detectioncell proliferation, Click-iT, flow cytometry, immunophenotyping
Scientific poster (2013) Improved click chemistry demonstrating EdU cell proliferation with GFP-expressing cells and R-PE-based immunophenotyping Alexa Fluor, cell cycle, cell proliferation, Click-iT, flow cytometry assays, fluorescent dyes, fluorescent proteins, immunophenotyping
Scientific poster (2010) Proliferative and phenotypic characterization of human mesenchymal stem cells by flow cytometry and imagingAlexa Fluor, cell proliferation, cells, Click-iT, flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, immunofluorescence (IF), immunophenotyping, stem cell research, violet laser-excited reagents
Scientific poster (2009) A comparative study of 25 different CD4 conjugates Alexa Fluor, antibodies, fluorescent dyes, immunophenotyping, multicolor flow cytometry, nanocrystals, Qdot
Scientific poster (2009) A novel xeno-free and serum-free culture medium for the expansion of human mesenchymal stem cells cell culture, cell expansion, flow cytometry, fluorescent dyes, gene expression, immunophenotyping, stem cell research
Scientific poster (2009) A simple, fast and quantitative single-step dead-cell indicator for flow cytometry apoptosis, fluorescent dyes, multicolor flow cytometry, nucleic acid quantitation, viability
Scientific poster (2009) Dead cell stains in flow cytometry: A comprehensive analysis apoptosis, cells, flow cytometry, fluorescent dyes, immunophenotyping, viability
Scientific poster (2009) Proliferative and phenotypic characterization of human mesenchymal stem cells by flow cytometry Alexa Fluor, cell proliferation, cells, Click-iT, flow cytometry, fluorescent dyes, fluorescent proteins, immunophenotyping, nucleic acid quantitation, stem cell research, violet laser-excited reagents
Scientific poster (2008) Zenon labeling technology facilitates detection of intra-cellular phosphoprotein determinants Alexa Fluor, antibodies, cell signaling, cells, flow cytometry, fluorescent dyes, protein labeling, rare cell detection, Zenon
Selection guideAntibody fixation considerationssample preparation
Selection guideHuman antibody cross reactivity chartantibody cross-reactivity, flow cytometry
Selection guideMouse antibody cross reactivity chartantibody cross-reactivity, flow cytometry
Selection guideCytokine bioassays guidecytokines, flow cytometry, immunophenotyping, intracellular staining
Selection guideIntracellular staining guide (for mouse & human cytokines and non-cytokine proteins)cytokines, flow cytometry, intracellular staining
Selection guideHuman CD & other cellular antigensflow cytometry, immunology, immunophenotyping, extracellular staining
Selection guideIntracellular staining buffer selection guideflow cytometry, immunophenotyping, intracellular staining
Selection guideMouse CD & other cellular antigensflow cytometry, immunology, immunophenotyping, extracellular staining
Selection guidePhospho flow cytometry antibody clone buffer selection guideflow cytometry, immunophenotyping, intracellular staining, phosphoflow cytometry
CYToMORE lecture video (2016) Assessing immunological aging using flow cytometry 
Presented by Victor Appay, Faculté de Médecine Pierre et Marie Curie, Pitié-Salpêtrière, France
flow cytometry, immunology, immunophenotyping
CYToMORE lecture video (2016) Cytometric analysis of cytokines and transcription factors 
Presented by Hyun-Dong Chang, Deutsches Rheuma-Forschungszentrum, Berlin, Germany
cell signaling, flow cytometry, immunophenotyping
CYToMORE lecture video (2016) Heterogeneity of tumour-infiltrating leukocytes: implications for adoptive cell transfer immunotherapy 
Presented by Enrico Lugli, Humanitas Clinical and Research Center, Italy
flow cytometry, immunology, immunophenotyping
CYToMORE lecture video (2016) Mass cytometry in the phenomics era: an essential tool for gene/function studies 
Presented by Herve Luche, Centre d’Immunophénomique - CIPHE, France
immunology, immunophenotyping, mass cytometry
How-to videoTutorial: Labeling a purified antibody 
Educational video on how to label your next antibody for imaging or fluorescence. Walk through the tutorial as Molecular Probes scientists demonstrate the protocol—including all the tips and tricks you'll want to know about for your next antibody labeling experiment. The video features Judie showing Curtis, a chemistry graduate student, how to label his monoclonal antibodies while saving time and maximizing yield.
antibodies, antibody labeling, flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes
How-to videoHow to site specifically label your antibody using SiteClick technology 
This video explains how to easily and site specifically label an antibody using an enzymatic and Click chemistry approach. This method can be applied to any intact IgG antibody and requires no antibody engineering or complex methodology. Unlike classic antibody conjugation techniques this breakthrough SiteClick technology allows for antibody conjugation with complete confidence that the label will not directly interfere with the antibody binding domain.
antibodies, antibody labeling, flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, Qdot, western detection
WebinarA practical approach to antibody labeling 
The growing number of fluorophores available makes labeling your own antibodies a tempting proposition. But with many antibody labeling solutions available, selecting the best option can be a daunting task. In this webinar we will:
  • Provide an overview of our antibody labeling kits
  • Offer guidance on which methods are ideal for specific applications and experiments
  • Provide tips and tricks to optimize your labeling protocol
antibodies, antibody labeling, flow cytometry, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF)
WebinarLearn to choose the right fluorophore when designing experiments 
The choice of fluorophore is one of the first important decisions to make in developing an experiment. Fluorophores are compounds that emit light at a specific wavelength when they have been excited at another, lower wavelength. In this webinar we will explore:
  • How to choose the best organic dye for an assay
  • Quantum dots and how they compare to other dyes
  • When to use a phycobiliprotein like R-PE or APC
  • When to use fluorescent proteins like GFP
  • How to choose a suitable dye to match your instrument
In addition, we will review the basic characteristics, strengths, and weaknesses of the various fluorophores to help you choose and develop the best assay for your needs.
antibodies, antibody labeling, flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, fluorescent proteins

Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.