IC Staining Flow Cytometry Protocol Using Detergents: R&D Systems

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class="container"> <div class="row"> <div id="protocol_menu" class="col-xs-12 col-md-3"> <div class="mobile_facet_wrapper"> <div class="search_mobile_buttons"> <div class="protocol_toggle btn btn-tertiary">Back to Protocols </div> </div> <div class="region region-left-sidebar"> <section id="block-views-protocols-menu-block" class="block block-views clearfix left-menu-wrapper"> <div class="left-menu"> <h5 class="menu-title">Protocols</h5> <div class="view view-Protocols-Menu view-id-Protocols_Menu view-display-id-block view-dom-id-063adfa98141b7439bcdc82a2b1e20ee"> <div class="view-content"> <div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Apoptosis Assays</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/protocols/recombinant-mouse-caspase-8-cleaved-bid-cytochrome-c-release-assay"><span>Recombinant Mouse Caspase-8-cleaved BID Cytochrome c Release Assay</span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/recombinant-mouse-bid-cytochrome-c-release-assay"><span>Recombinant Mouse BID Cytochrome c Release Assay</span></a> </div></li> <li class="views-row views-row-3 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/recombinant-mouse-bcl-xl-cytochrome-c-release-assay"><span>Recombinant Mouse Bcl-xL Cytochrome c Release Assay</span></a> </div></li> <li class="views-row views-row-4 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-caspase-8-cleaved-bid-cytochrome-c-release-assay"><span>Recombinant Human Caspase-8-cleaved BID Cytochrome c Release Assay</span></a> </div></li> <li class="views-row views-row-5 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-bim-l-cytochrome-c-release-assay"><span>Recombinant Human BIM-L Cytochrome c Release Assay</span></a> </div></li> <li class="views-row views-row-6 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-bid-cytochrome-c-release-assay"><span>Recombinant Human BID Cytochrome c Release Assay</span></a> </div></li> <li class="views-row views-row-7 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-bcl-xl-cytochrome-c-release"><span>Recombinant Human Bcl-xL Cytochrome c Release </span></a> </div></li> <li class="views-row views-row-8 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-bcl-x"><span>Recombinant Human Bcl-x</span></a> </div></li> <li class="views-row views-row-9 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-bcl-2-cytochrome-c-release-assay"><span>Recombinant Human Bcl-2 Cytochrome c Release Assay</span></a> </div></li> <li class="views-row views-row-10 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/preparation-samples-cytochrome-c-elisa"><span>Preparation of the Samples for Cytochrome c ELISA</span></a> </div></li> <li class="views-row views-row-11 views-row-odd views-row-last"> <div class="menu-child"> <a href="/resources/protocols/caspase-activity-assays-inhibitors-apoptosis-mitochondrial-proteins"><span>Caspase Activity Assays for Inhibitors of Apoptosis & Mitochondrial Proteins</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Enzyme Assays</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/technical/sulfotransferase-activity-assays"><span>Sulfotransferase Activity Assays</span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/recombinant-mouse-heparan-sulfate-6-o-sulfotransferase-3hs6st3-activity-assay"><span>Recombinant Mouse Heparan Sulfate 6-O-Sulfotransferase-3/HS6ST3 Activity Assay</span></a> </div></li> <li class="views-row views-row-3 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/recombinant-mouse-carbohydrate-sulfotransferase-7chst7-activity-assay"><span>Recombinant Mouse Carbohydrate Sulfotransferase 7/CHST7 Activity Assay</span></a> </div></li> <li class="views-row views-row-4 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/recombinant-mouse-carbohydrate-sulfotransferase-5chst5-activity-assay"><span>Recombinant Mouse Carbohydrate Sulfotransferase 5/CHST5 Activity Assay</span></a> </div></li> <li class="views-row views-row-5 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/recombinant-mouse-carbohydrate-sulfotransferase-1chst1-activity-assay"><span>Recombinant Mouse Carbohydrate Sulfotransferase 1/CHST1 Activity Assay</span></a> </div></li> <li class="views-row views-row-6 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/recombinant-mouse-ace-2-assay"><span>Recombinant Mouse ACE-2 Assay</span></a> </div></li> <li class="views-row views-row-7 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-sulfate-6-o-sulfotransferase-1hs6st1-activity-assay"><span>Recombinant Human Sulfate 6-O-Sulfotransferase 1/HS6ST1 Activity Assay</span></a> </div></li> <li class="views-row views-row-8 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-meprin-alpha-subunitmep1a-assay"><span>Recombinant Human Meprin alpha Subunit/MEP1A Assay</span></a> </div></li> <li class="views-row views-row-9 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-galnac4s-6stbrag-activity-assay"><span>Recombinant Human GalNAc4S-6ST/BRAG Activity Assay</span></a> </div></li> <li class="views-row views-row-10 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-carbohydrate-sulfotransferase-6chst6-activity-assay"><span>Recombinant Human Carbohydrate Sulfotransferase 6/CHST6 Activity Assay</span></a> </div></li> <li class="views-row views-row-11 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-carbohydrate-sulfotransferase-4chst4-activity-assay"><span>Recombinant Human Carbohydrate Sulfotransferase 4/CHST4 Activity Assay</span></a> </div></li> <li class="views-row views-row-12 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-carbohydrate-sulfotransferase-2chst2-activity-assay"><span>Recombinant Human Carbohydrate Sulfotransferase 2/CHST2 Activity Assay</span></a> </div></li> <li class="views-row views-row-13 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-carbohydrate-sulfotransferase-1chst1-activity-assay"><span>Recombinant Human Carbohydrate Sulfotransferase 1/CHST1 Activity Assay</span></a> </div></li> <li class="views-row views-row-14 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-bmp-1pcp-assay"><span>Recombinant Human BMP-1/PCP Assay</span></a> </div></li> <li class="views-row views-row-15 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/recombinant-human-ace-2-assay"><span>Recombinant Human ACE-2 Assay</span></a> </div></li> <li class="views-row views-row-16 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/inhibition-calcineurin-phosphatase-activity"><span>Inhibition of Calcineurin Phosphatase Activity</span></a> </div></li> <li class="views-row views-row-17 views-row-odd views-row-last"> <div class="menu-child"> <a href="/resources/protocols/enzyme-activity-assays-using-fluorogenic-peptide-substrates"><span>Enzyme Activity Assays using Fluorogenic Peptide Substrates</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 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class="menu-child"> <a href="/resources/protocols/analysis-cell-viability-using-7-amino-actinomycin-d-7-aad"><span>7-Amino Actinomycin D (7-AAD) Cell Viability Flow Cytometry Protocol</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">ICC/IHC Protocols</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/protocols/importance-ihcicc-controls"><span>The Importance of IHC/ICC Controls</span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/protocol-visucyte-hrp-polymer-detection-reagent"><span>Protocol for VisUCyte™ HRP Polymer Detection Reagent</span></a> </div></li> <li class="views-row views-row-3 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/protocol-preparation-gelatin-coated-slides-histological-tissue-sections"><span>Protocol for the Preparation of Gelatin-coated Slides for Histological Tissue Sections</span></a> </div></li> <li class="views-row views-row-4 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/protocol-preparation-fixation-cells-coverslips"><span>Protocol for the Preparation & Fixation of Cells on Coverslips</span></a> </div></li> <li class="views-row views-row-5 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/protocol-making-4-formaldehyde-solution-pbs"><span>Protocol for Making a 4% Formaldehyde Solution in PBS</span></a> </div></li> <li class="views-row views-row-6 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/protocol-heat-induced-epitope-retrieval-hier"><span>Protocol for Heat-Induced Epitope Retrieval (HIER)</span></a> </div></li> <li class="views-row views-row-7 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/primary-antibody-selection-optimization"><span>Primary Antibody Selection & Optimization</span></a> </div></li> <li class="views-row views-row-8 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/preventing-non-specific-staining"><span>Preventing Non-specific Staining</span></a> </div></li> <li class="views-row views-row-9 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/preparing-samples-ihcicc-experiments"><span>Preparing Samples for IHC/ICC Experiments</span></a> </div></li> <li class="views-row views-row-10 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/detection-visualization-antibody-binding"><span>Detection & Visualization of Antibody Binding</span></a> </div></li> <li class="views-row views-row-11 views-row-odd"> <div class="menu-child"> <a href="/resources/technical/designing-successful-ihc-icc-experiment"><span>Designing a Successful IHC/ICC Experiment</span></a> </div></li> <li class="views-row views-row-12 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/appropriate-fixation-ihcicc-samples"><span>Appropriate Fixation of IHC/ICC Samples</span></a> </div></li> <li class="views-row views-row-13 views-row-odd views-row-last"> <div class="menu-child"> <a href="/resources/protocols/antigen-retrieval-methods"><span>Antigen Retrieval Methods</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Immunocytochemistry (ICC) Protocols</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-stem-cells-coverslips"><span>Protocol for the Preparation and Fluorescent ICC Staining of Stem Cells on Coverslips</span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-non-adherent-cells"><span>Protocol for the Preparation and Fluorescent ICC Staining of Non-adherent Cells</span></a> </div></li> <li class="views-row views-row-3 views-row-odd views-row-last"> <div class="menu-child"> <a href="/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-cells-coverslips"><span>Protocol for the Preparation and Fluorescent ICC Staining of Cells on Coverslips</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Immunohistochemistry (IHC) Protocols</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/technical/troubleshooting-guide-immunohistochemistry"><span>Troubleshooting Guide: Immunohistochemistry</span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-paraffin-embedded-tissue"><span>Protocol for the Preparation and Fluorescent IHC Staining of Paraffin-embedded Tissue Sections</span></a> </div></li> <li class="views-row views-row-3 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/protocol-preparation-and-chromogenic-ihc-staining-paraffin-embedded-tissue"><span>Protocol for the Preparation and Chromogenic IHC Staining of Paraffin-embedded Tissue Sections</span></a> </div></li> <li class="views-row views-row-4 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-frozen-tissue-sections"><span>Protocol for the Preparation and Fluorescent IHC Staining of Frozen Tissue Sections</span></a> </div></li> <li class="views-row views-row-5 views-row-odd views-row-last"> <div class="menu-child"> <a href="/resources/protocols/protocol-preparation-and-chromogenic-ihc-staining-frozen-tissue-sections"><span>Protocol for the Preparation and Chromogenic IHC Staining of Frozen Tissue Sections</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Immunology</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/protocols/protocol-differentiation-and-characterization-human-th2-cells"><span>Protocol for the Differentiation and Characterization of Human Th2 Cells</span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/protocol-differentiation-and-characterization-human-th17-cells"><span>Protocol for the Differentiation and Characterization of Human Th17 Cells</span></a> </div></li> <li class="views-row views-row-3 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/protocol-differentiation-and-characterization-human-th1-cells"><span>Protocol for the Differentiation and Characterization of Human Th1 Cells</span></a> </div></li> <li class="views-row views-row-4 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/protocol-characterization-human-th9-cells"><span>Protocol for the Characterization of Human Th9 Cells</span></a> </div></li> <li class="views-row views-row-5 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/protocol-characterization-human-th22-cells"><span>Protocol for the Characterization of Human Th22 Cells</span></a> </div></li> <li class="views-row views-row-6 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/protocol-characterization-human-regulatory-t-cells"><span>Protocol for the Characterization of Human Regulatory T Cells</span></a> </div></li> <li class="views-row views-row-7 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/cd4-t-cell-isolation-pbmc-or-splenocyte-preparations-t-cell-enrichment-column"><span>CD4+ T Cell Isolation from PBMC or Splenocyte Preparations by T Cell Enrichment Column</span></a> </div></li> <li class="views-row views-row-8 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/cd4-t-cell-isolation-pbmc-or-splenocyte-preparations-magnetic-selection"><span>CD4+ T Cell Isolation from PBMC or Splenocyte Preparations by Magnetic Selection</span></a> </div></li> <li class="views-row views-row-9 views-row-odd views-row-last"> <div class="menu-child"> <a href="/resources/protocols/cd4-cd25-regulatory-t-cell-isolation-pbmc-or-splenocyte-preparations-magnetic"><span>CD4+ CD25+ Regulatory T Cell Isolation from PBMC or Splenocyte Preparations by Magnetic Selection</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Luminex</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first views-row-last"> <div class="menu-child"> <a href="/resources/protocols/troubleshooting-guide-luminex"><span>Luminex® Troubleshooting Guide</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Neural Cell Culturing</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/protocols/protocol-culturing-rat-microglia"><span>Protocol for Culturing Rat Microglia</span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/protocol-culturing-rat-hippocampal-neurons"><span>Protocol for Culturing Rat Hippocampal Neurons</span></a> </div></li> <li class="views-row views-row-3 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/protocol-culturing-rat-cortical-neurons"><span>Protocol for Culturing Rat Cortical Neurons</span></a> </div></li> <li class="views-row views-row-4 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/protocol-culturing-embryonic-rat-spinal-motor-neurons"><span>Protocol for Culturing Embryonic Rat Spinal Motor Neurons</span></a> </div></li> <li class="views-row views-row-5 views-row-odd views-row-last"> <div class="menu-child"> <a href="/resources/protocols/protocol-culturing-embryonic-chick-dorsal-root-ganglion-neurons"><span>Protocol for Culturing Embryonic Chick Dorsal Root Ganglion Neurons</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Organoid and 3-D Cell Culture Protocols</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/protocols/use-cultrex-basement-membrane-extract"><span>Use of Cultrex Basement Membrane Extract to Improve Take and Growth of Xenografts in Mice </span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/immunofluorescence-organoids-embedded-cultrex-basement"><span>Immunofluorescence of Organoids Embedded in Cultrex Basement Membrane Extract </span></a> </div></li> <li class="views-row views-row-3 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/human-lung-organoid-culture-protocol"><span>Human Lung Organoid Culture Protocol</span></a> </div></li> <li class="views-row views-row-4 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/human-liver-organoid-culture-protocol"><span>Human Liver Organoid Culture Protocol</span></a> </div></li> <li class="views-row views-row-5 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/human-intestinal-organoid-culture-protocol"><span>Human Intestinal Organoid Culture Protocol</span></a> </div></li> <li class="views-row views-row-6 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/harvesting-organoids-biochemical-analysis"><span>Harvesting Organoids for Biochemical Analysis </span></a> </div></li> <li class="views-row views-row-7 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/culture-normal-human-gastric-organoids"><span>Culture of Normal Human Gastric Organoids </span></a> </div></li> <li class="views-row views-row-8 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/culture-mouse-enteric-organoids-using-cultrex-basement"><span>Culture of Mouse Enteric Organoids using Cultrex Basement Membrane Extract</span></a> </div></li> <li class="views-row views-row-9 views-row-odd views-row-last"> <div class="menu-child"> <a href="/resources/protocols/cultrex-ultimatrix-bme-protocol"><span>Cultrex UltiMatrix BME Protocol</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Proteome Profiler Arrays</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/protocols/li-cor-data-examples-rat-cytokine-array-panel%20a"><span>LI-COR Data Examples for Rat Cytokine Array Panel A</span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/li-cor-data-examples-human-angiogenesis-array-kit"><span>LI-COR Data Examples for Human Angiogenesis Array Kit</span></a> </div></li> <li class="views-row views-row-3 views-row-odd views-row-last"> <div class="menu-child"> <a href="/resources/protocols/li-cor-data-exmaples-human-adipokine-array-kit"><span>LI-COR Data Examples for Human Adipokine Array Kit</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Stem Cell Protocols</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/protocols/mouse-pre-b-colony-forming-cell-cfc-assay-using-methylcellulose-based-media"><span>The Mouse Pre-B Colony Forming Cell (CFC) Assay Using Methylcellulose-based Media</span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/mouse-colony-forming-cell-cfc-assay-using-methylcellulose-based-media"><span>The Mouse Colony Forming Cell (CFC) Assay using Methylcellulose-based Media</span></a> </div></li> <li class="views-row views-row-3 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/human-colony-forming-cell-cfc-assay-using-methylcellulose-based-media"><span>The Human Colony Forming Cell (CFC) Assay using Methylcellulose-based Media</span></a> </div></li> <li class="views-row views-row-4 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/rat-cortical-stem-cell-culture-expansion-protocol"><span>Rat Cortical Stem Cell Culture Expansion Protocol</span></a> </div></li> <li class="views-row views-row-5 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/protocol-preparation-fixation-stem-cells-coverslips"><span>Protocol for the Preparation & Fixation of Stem Cells on Coverslips</span></a> </div></li> <li class="views-row views-row-6 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/procedure-expansion-human-mesenchymal-stem-cells"><span>Procedure for the Expansion of Human Mesenchymal Stem Cells</span></a> </div></li> <li class="views-row views-row-7 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/procedure-serum-free-expansion-human-mesenchymal-stem-cells"><span>Procedure for Serum-Free Expansion of Human Mesenchymal Stem Cells</span></a> </div></li> <li class="views-row views-row-8 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/osteogenic-differentiation-humanmouse-mesenchymal-stem-cells"><span>Osteogenic Differentiation of Human/Mouse Mesenchymal Stem Cells</span></a> </div></li> <li class="views-row views-row-9 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/mouse-hematopoietic-lineage-depletion"><span>Mouse Hematopoietic Lineage Depletion</span></a> </div></li> <li class="views-row views-row-10 views-row-even"> <div class="menu-child"> <a href="/resources/technical/endoderm-ectoderm-and-mesoderm-differentiation-bg01v-human-embryonic-stem-cells"><span>Endoderm, Ectoderm, and Mesoderm Differentiation of BG01V Human Embryonic Stem Cells</span></a> </div></li> <li class="views-row views-row-11 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/definitive-endoderm-differentiation-bg01v-human-embryonic-stem-cells"><span>Definitive Endoderm Differentiation of BG01V Human Embryonic Stem Cells</span></a> </div></li> <li class="views-row views-row-12 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/culturing-rat-cortical-stem-cells-expansion-using-neurosphere-system"><span>Culturing Rat Cortical Stem Cells: Expansion using the Neurosphere System</span></a> </div></li> <li class="views-row views-row-13 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/culturing-rat-cortical-stem-cells-expansion-using-monolayer-system"><span>Culturing Rat Cortical Stem Cells: Expansion using the Monolayer System</span></a> </div></li> <li class="views-row views-row-14 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/culturing-mouse-cortical-stem-cells-expansion-using-neurosphere-system"><span>Culturing Mouse Cortical Stem Cells: Expansion using the Neurosphere System</span></a> </div></li> <li class="views-row views-row-15 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/culturing-bg01v-human-embryonic-stem-cells-mouse-embryonic-fibroblast-mef"><span>Culturing BG01V Human Embryonic Stem Cells with Mouse Embryonic Fibroblast (MEF)-Conditioned Media</span></a> </div></li> <li class="views-row views-row-16 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/culturing-bg01v-human-embryonic-stem-cells-irradiated-mouse-embryonic"><span>Culturing BG01V Human Embryonic Stem Cells on Irradiated Mouse Embryonic Fibroblasts (iMEF)</span></a> </div></li> <li class="views-row views-row-17 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/chondrogenic-differentiation-humanmouse-mesenchymal-stem-cells"><span>Chondrogenic Differentiation of Human/Mouse Mesenchymal Stem Cells</span></a> </div></li> <li class="views-row views-row-18 views-row-even views-row-last"> <div class="menu-child"> <a href="/resources/protocols/adipogenic-differentiation-humanmouse-mesenchymal-stem-cells"><span>Adipogenic Differentiation of Human/Mouse Mesenchymal Stem Cells</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Ubiquitin</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/protocols/vitro-ubiquitin-conjugation-reaction"><span>Protocol for In Vitro Ubiquitin Conjugation Reaction</span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/distinguish-between-poly-ubiquitination-and-multi-mono-ubiquitination"><span>Distinguish Between Poly-ubiquitination and Multi-mono-ubiquitination</span></a> </div></li> <li class="views-row views-row-3 views-row-odd views-row-last"> <div class="menu-child"> <a href="/resources/protocols/determine-ubiquitin-chain-linkage"><span>Determine Ubiquitin Chain Linkage</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Video Protocols</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/protocols/profiling-changes-receptor-tyrosine-kinase-phosphorylation-using-antibody-arrays"><span>Profiling Changes in Receptor Tyrosine Kinase Phosphorylation using Antibody Arrays </span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/induction-and-analysis-epithelial-mesenchymal-transition"><span>Induction and Analysis of Epithelial to Mesenchymal Transition</span></a> </div></li> <li class="views-row views-row-3 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/how-run-cometassay-analysis-software"><span>How to Run the CometAssay Analysis Software</span></a> </div></li> <li class="views-row views-row-4 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/how-run-r-and-d-systems-quantikine-quickit-elisa"><span>How to Run an R&D Systems Quantikine® QuicKit™ ELISA</span></a> </div></li> <li class="views-row views-row-5 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/how-run-rd-systems-quantikine-elisa"><span>How to Run an R&D Systems Quantikine ELISA</span></a> </div></li> <li class="views-row views-row-6 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/how-run-rd-systems-luminex-assay"><span>How to Run an R&D Systems Luminex® Assay</span></a> </div></li> <li class="views-row views-row-7 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/how-run-rd-systems-duoset-elisa"><span>How to Run an R&D Systems DuoSet ELISA</span></a> </div></li> <li class="views-row views-row-8 views-row-even"> <div class="menu-child"> <a href="/resources/protocols/how-reconstitute-lyophilized-proteins"><span>How to Reconstitute Lyophilized Proteins</span></a> </div></li> <li class="views-row views-row-9 views-row-odd"> <div class="menu-child"> <a href="/resources/protocols/flow-cytometry-protocol-staining-membrane-associated-proteins"><span>Flow Cytometry Protocol for Staining Membrane Associated Proteins</span></a> </div></li> <li class="views-row views-row-10 views-row-even views-row-last"> <div class="menu-child"> <a href="/resources/protocols/guide-colony-forming-cell-assay-methods-and-tips"><span>A Guide to the Colony Forming Cell Assay: Methods and Tips</span></a> </div></li> </ul></div><div class="item-list"> <div class="menu-title-wrap"> <h5 class="menu-subtitle">Western Blot</h5> </div> <ul> <li class="views-row views-row-1 views-row-odd views-row-first"> <div class="menu-child"> <a href="/resources/protocols/western-blot-cell-lysate-protocol"><span>Western Blot Protocol for Cell Lysates</span></a> </div></li> <li class="views-row views-row-2 views-row-even"> <div class="menu-child"> <a href="/resources/technical/western-blot-conditions"><span>Western Blot Conditions</span></a> </div></li> <li class="views-row views-row-3 views-row-odd"> <div class="menu-child"> <a href="/resources/technical/troubleshooting-guide-western-blot-figures"><span>Troubleshooting Guide: Western Blot Figures</span></a> </div></li> <li class="views-row views-row-4 views-row-even"> <div class="menu-child"> <a href="/resources/technical/troubleshooting-guide-western-blot"><span>Troubleshooting Guide: Western Blot</span></a> </div></li> <li class="views-row views-row-5 views-row-odd views-row-last"> <div class="menu-child"> <a href="/resources/protocols/western-blot-qc-protocol"><span>R&D Systems Quality Control Western Blot Protocol</span></a> </div></li> </ul></div> </div> </div> </div> </section> </div> </div> </div> <div id="protocol_right" class="col-xs-12 col-md-9"> <div class="search_mobile_buttons"> <div class="protocol_menu_toggle btn btn-tertiary">Show Categories</div> </div> <div class="region region-content"> <section id="block-system-main" class="block block-system clearfix"> <article id="node-3496" class="node node-protocols clearfix" about="/resources/protocols/flow-cytometry-protocol-staining-intracellular-molecules-using-detergents" typeof="sioc:Item foaf:Document"> <header> <span property="dc:title" content="Intracellular Staining Flow Cytometry Protocol Using Detergents to Permeabilize Cells" class="rdf-meta element-hidden"></span><span property="sioc:num_replies" content="0" datatype="xsd:integer" class="rdf-meta element-hidden"></span> </header> <div class="field field-name-body field-type-text-with-summary field-label-hidden"><div class="field-items"><div class="field-item even" property="content:encoded"><div class="protocols"> <h3>Intracellular Flow Cytometry Overview</h3> <div class="row"> <div class="col-md-7 col-xs-12"> <p>Flow cytometry can be used to analyze various intracellular molecules including phosphorylated signaling proteins and cytokines. To stain intracellular molecules, the cells need to be fixed in suspension and then permeabilized before the detection antibody is added. This fixation/permeabilization treatment allows the antibody to pass through the plasma membrane into the cell interior, while maintaining the morphological characteristics used to sort the cells. Commonly used detergents include saponin, Triton<sup>Æ</sup> X-100, or Tween<sup>Æ</sup> 20.</p> <p>The following flow cytometry staining protocol has been developed and optimized by R&D Systems Flow Cytometry Laboratory. This protocol is designed for intracellular staining of proteins. It is recommended that experimental conditions, such as antibody concentration, incubation time, and temperature, be optimized for each flow cytometry experiment.</p> <p><em>Please read the following flow cytometry staining protocol in its entirety before beginning.</em></p> </div> <div class="col-md-5 col-xs-12"><img alt="Intracellular Flow Cytometry Staining" src="//resources.rndsystems.com/images/intracellular-flow-stain-335px4.png" width="100%" /></div> </div>  <div class="row"> <div class="col-xs-12"> <h3>Contents</h3> <ul> <li><a href="#methods">Required Reagents and Materials</a></li> <li><a href="#procedure">Procedure</a></li> <li><a href="#example">Data Example</a></li> </ul> </div> </div> <div class="row protocols-methods"> <div class="col-xs-12"> <h3 id="methods" name="methods">Reagents Required</h3> <ul> <li>PBS (1X): 0.137 M NaCl, 0.05 M NaH<sub>2</sub>PO<sub>4</sub>, pH 7.4 or Hank's Balanced Salt Solution (HBSS; 1X)</li> <li><a href="/products/flow-cytometry-fixation-buffer-1x-_fc004">Flow Cytometry Fixation Buffer</a> (Catalog # FC004) or an equivalent solution containing 1 - 4% paraformaldehyde</li> <li><a href="/products/flow-cytometry-permeabilization-wash-buffer-i-1x-_fc005">Flow Cytometry Permeabilization Buffer/Wash Buffer I</a> (Catalog # FC005) or an equivalent solution containing a permeabilization agent such as saponin or Triton X-100</li> <li>(Optional, see Note in step 3) <a href="/products/flow-cytometry-fixation-permeabilization-buffer-i-1x-_fc007">Flow Cytometry Fixation/Permeabilization Buffer I</a> (Catalog # FC007)</li> <li>Fc Receptor Blocking Reagents (These include Fc receptor blocking antibodies or IgG solutions)</li> <li><a href="/products/conjugated-primary-antibodies">Fluorochrome-conjugated antibodies</a> suitable for use in flow cytometry</li> <li><a href="/search?keywords=isotype&category=Isotype%20Controls">Isotype Control Antibodies</a></li> </ul> <h3>Materials Required</h3> <ul> <li>FACS<sup>ô</sup> Tubes (5 mL round-bottom polystyrene tubes)</li> <li>Pipette Tips and Pipettes</li> <li>Centrifuge</li> <li>Vortex</li> </ul> </div> </div> <div class="row"> <div class="col-xs-12" itemprop="step" itemscope="" itemtype="http://schema.org/HowToSection"> <h3 id="Intracellular Staining Flow Cytometry Protocol Using Detergents to Permeabilize Cells" itemprop="name">Procedure</h3> <p>When performing surface and intracellular staining in the same sample, it is advisable that the <a href="/resources/protocols/flow-cytometry-protocol-staining-membrane-associated-proteins-suspended-cells">staining of cell surface antigens</a> be performed first since fixation and permeabilization treatments might decrease the availability of surface antigens.</p> <div itemprop="itemListElement" itemscope="" itemtype="http://schema.org/HowToDirection"><meta itemprop="url" content="https://www.rndsystems.com/resources/protocols/flow-cytometry-protocol-staining-intracellular-molecules-using-detergents"/> <ol><meta itemprop="position" content="1"/> <li itemprop="text">Harvest cells and wash 2 times by adding 2 mL of PBS (or HBSS), centrifuging at 1250-1500 rpm/350-500 x g for 5 minutes, and then decanting buffer from pelleted cells.</li> <br /> <meta itemprop="position" content="2"/> <li itemprop="text">Aliquot up to 1 x 10<sup>6</sup> cells/100 µL into FACS tubes. Add 0.5 mL of cold <a href="/products/flow-cytometry-fixation-buffer-1x_fc004">Flow Cytometry Fixation Buffer</a> (Catalog # FC004) and vortex. Incubate at room temperature for 10 minutes. Vortex cells intermittently in order to maintain a single cell suspension.</li> <br /> <meta itemprop="position" content="3"/> <li itemprop="text">Centrifuge cells and decant the Fixation Buffer. Wash cells 2 times with PBS (or HBSS) as described in step 1. Resuspend the cell pellet in 100 ñ 200 µL of <a href="/products/flow-cytometry-permeabilization-wash-buffer-i-1x-_fc005">Flow Cytometry Permeabilization Buffer/Wash Buffer I</a> (Catalog # FC005)<br /> <span class="noteem"><em><strong>Note:</strong> Saponin-mediated cell permeabilization is a reversible process, it is important to keep the cells in the presence of Permeabilization Buffer I during intracellular staining.</em></span><br /> <span class="noteem"><em><strong>Note:</strong> Depending on the specific antibody and cell sample being used, the fixation and permeabilization steps can be performed simultaneously using <a href="/products/flow-cytometry-fixation-permeabilization-buffer-i-1x-_fc007">Flow Cytometry Fixation/Permeabilization Buffer I </a>(Catalog # FC007).</em></span></li> <br /> <meta itemprop="position" content="4"/> <li itemprop="text">Fc-block cells with blocking IgG (1 µg IgG/10<sup>6</sup> cells) for 15 minutes at room temperature.<br /> <span class="noteem"><em><strong>Note:</strong> Do not wash excess blocking IgG from this reaction.</em></span></li> <br /> <meta itemprop="position" content="5"/> <li itemprop="text">Add conjugated antibody (5-10 µL/10<sup>6</sup> cells or a previously titrated amount) and vortex. Incubate cells for 30 minutes at room temperature in the dark.</li> <br /> <meta itemprop="position" content="6"/> <li itemprop="text">Wash cells 2 times with <a href="/products/flow-cytometry-permeabilization-wash-buffer-i-1x-_fc005">Flow Cytometry Permeabilization Buffer/Wash Buffer I</a> (Catalog # FC005) as described in step 1.<br /> <span class="noteem"><em><strong>Note:</strong> If an unconjugated primary antibody is used, incubation with an appropriate <a href="/search?keywords=Secondary%20Antibodies&category=Secondary%20Antibodies&rnd_applications=Flow%20Cytometry">secondary antibody</a> should occur now. Dilute the secondary antibody in PBS (or HBSS), starting with the concentration suggested in the product datasheet. Incubate for 20-30 minutes in the dark and wash as described in step 1.</em></span></li> <br /> <meta itemprop="position" content="7"/> <li itemprop="text">Resuspend the cells in 200 ñ 400 µL PBS (or HBSS) buffer for flow cytometric analysis.</li> </ol> <p>It is recommended to run a negative control. A separate set of cells should be stained with an <a href="/search?keywords=isotype&category=Isotype%20Controls">Isotype Control Antibody</a> using the steps outlined above.</p> <p>FACS is a trademark of Becton Dickinson and Company.<br /> Triton is a registered trademark of The Dow Chemical Company.<br /> Tween is a registered trademark of Uniqema Americas LLC.</p> </div> </div> </div>  <div class="row"> <div class="col-xs-12"> <h3 id="example" name="example">Flow Cytometry Intracellular Staining Example</h3> </div> <div class="col-md-5 col-xs-12"><img alt="Detection of CCL4/MIP 1 beta in Human Blood Monocytes by Flow Cytometry." src="//resources.rndsystems.com/images/site/detection-CCL4-MIP-1.png" width="450px" /></div> <div class="col-md-7 col-xs-12"><strong>Detection of CCL4/MIP 1 beta in Human Blood Monocytes by Flow Cytometry.</strong> Human peripheral blood monocytes treated with LPS and Monensin for 5 hours were stained with an <a href="/products/human-cd14-apc-conjugated-antibody-134620_fab3832a">APC-conjugated Mouse Anti-Human CD14  Monoclonal Antibody</a> (Catalog # FAB3832A) and either (A) <a href="/products/human-ccl4-mip-1beta-fluorescein-conjugated-antibody-24006_ic271f">Fluorescein-conjugated Mouse Anti-Human CCL4/MIP 1 beta Monoclonal Antibody</a> (Catalog # IC271F) or (B) <a href="/products/mouse-igg2b-fluorescein-conjugated-control-antibody_ic0041f">Fluorescein-conjugated Mouse IgG2B Isotype Control</a> (Catalog # IC0041F). To facilitate intracellular staining, cells were fixed with <a href="/products/flow-cytometry-fixation-buffer-1x_fc004">Flow Cytometry Fixation Buffer</a> (Catalog # FC004) and permeabilized with <a href="/products/flow-cytometry-permeabilization-wash-buffer-i-1x_fc005">Flow Cytometry Permeabilization/Wash Buffer I</a> (Catalog # FC005).</div> </div> </div> <div> <h3>Protocol Category</h3> </div> <style type="text/css">.protocols > .row {margin-top: 15px; margin-bottom: 15px;} .protocols-methods { background-color: #e0eaea; padding-top: 15px; padding-bottom: 15px;} .relatedcontent_links {margin-top:15px;} </style> </div></div></div><div class="field field-name-field-category field-type-taxonomy-term-reference field-label-hidden"><div class="field-items"><div class="field-item even"><a href="/protocol-types/flow-cytometry" typeof="skos:Concept" property="rdfs:label skos:prefLabel">Flow Cytometry</a></div></div></div> </article> </section> <section id="block-block-26" class="block 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block-et-messages clearfix"> <span></span> </section> <section id="block-et-relatedcontent-menu-et-relatedcontent-menu-block" class="block block-et-relatedcontent-menu clearfix"> <div class="relatedcontent_links"><h3>Related Information</h3></div> <div class="row evenheight"><div class="col-md-6 col-xs-12"><a href="https://www.rndsystems.com/resources/cell-markers" class="btn btn-senary btn-relatedinfo" itemprop="relatedLink">Cell Markers</a></div><div class="col-md-6 col-xs-12"><a href="https://www.rndsystems.com/resources/protocols/flow-cytometry-protocols" class="btn btn-senary btn-relatedinfo" itemprop="relatedLink">Flow Cytometry Protocols</a></div><div class="col-md-6 col-xs-12"><a href="https://www.rndsystems.com/products/flow-cytometry-supplemental-reagents" class="btn btn-senary btn-relatedinfo" itemprop="relatedLink">Flow Cytometry Supplemental reagents</a></div><div class="col-md-6 col-xs-12"><a href="https://www.rndsystems.com/product-highlights/flow-cytometry-training-webinars" class="btn btn-senary btn-relatedinfo" itemprop="relatedLink">Flow Cytometry Training Webinars</a></div><div class="col-md-6 col-xs-12"><a href="https://www.rndsystems.com/products/flow-cytometry-workflow-solutions" class="btn btn-senary btn-relatedinfo" itemprop="relatedLink">Flow Cytometry Workflow Solutions</a></div><div class="col-md-6 col-xs-12"><a href="https://www.rndsystems.com/resources/human-cd-marker-products" class="btn btn-senary btn-relatedinfo" itemprop="relatedLink">Human CD Marker Products</a></div></div> </section> <section id="block-et-recommendation-engine-et-recommendation-engine-block" class="block block-et-recommendation-engine clearfix"> <div class="recommendation-engine-wrapper recommendation-engine-margin-top" id="protocols"> <h3>Recommended Products</h3> <div class="owl-carousel-recommended-products owl-carousel owl-theme owl-loaded owl-drag"> <div class="owl-stage-outer"> 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Immunocytochemistry (ICC)." title="Cytokeratin 19 antibody in MCF-7 Human Cell Line by Immunocytochemistry (ICC)." class="b-lazy" loading="lazy"/> </div> </a></div> <div class="complete_title"> <a data-toggle="tooltip" data-placement="top" title="" href="https://www.rndsystems.com/products/human-cytokeratin-19-alexa-fluor-488-conjugated-antibody_ic3506g" class="font-weight-bold text-ellipsis safe_link recommended-product-link" data-link-type="title"> <strong>Human Cytokeratin 19 Alexa Fluor® 488-conjugated Antibody</strong> </a> </div> <div><strong>Cat #</strong> IC3506G</div> <div> <a href="https://www.rndsystems.com/products/human-cytokeratin-19-alexa-fluor-488-conjugated-antibody_ic3506g#product-citations" class="font-weight-bold safe_link recommended-product-link" data-link-type="citations"> <strong>Citations (1)</strong> </a> </div> </div> </div> </div> <div class="owl-item"> <div class="item"> <div class="recommended-product-container recommended-product" data-prod-brand="rnd" data-prod-category="Primary Antibodies" data-prod-catnum="MAB2400" data-prod-name="Human/Mouse/Rat HNF-3 beta /FoxA2 Antibody" data-prod-position="14"> <div><a href="https://www.rndsystems.com/products/human-mouse-rat-hnf-3beta-foxa2-antibody-2141a_mab2400" class="safe_link recommended-product-link" data-link-type="image"> <div class="img-border rounded p-2 item d-inline-block m___product-recommended w-100 text-center" data-toggle="tooltip" data-placement="top" title="Human/Mouse/Rat HNF-3 beta /FoxA2 Antibody"> <img src="https://resources.rndsystems.com/images/datasheets/antibody/HNF3_beta_MAB2400_Western_Blot_21151.jpg" data-src="https://resources.rndsystems.com/images/datasheets/antibody/HNF3_beta_MAB2400_Western_Blot_21151.jpg" alt="Detection of Human HNF-3 beta /FoxA2 antibody by Western Blot." title="Detection of Human HNF-3 beta /FoxA2 antibody by Western Blot." class="b-lazy" loading="lazy"/> </div> </a></div> <div class="complete_title"> <a data-toggle="tooltip" data-placement="top" title="" href="https://www.rndsystems.com/products/human-mouse-rat-hnf-3beta-foxa2-antibody-2141a_mab2400" class="font-weight-bold text-ellipsis safe_link recommended-product-link" data-link-type="title"> <strong>Human/Mouse/Rat HNF-3 beta /FoxA2 Antibody</strong> </a> </div> <div><strong>Cat #</strong> MAB2400</div> </div> </div> </div> </div> </div> </div> </div> </section> </div> </div> </div> </div> </div> <footer id="footer_wrapper" class="container-fluid"> <div id="footer_wrapper_container" class="row"> <div id="footer" class="container"> <div id="footer_container" class="row"> <nav class="footer_column footer_column_1 col-xs-12 col-sm-3"> <div class="region region-footer-column-1"> <section id="block-menu-menu-footer-popular-searches" class="block block-menu clearfix"> <h2 class="block-title">Popular Products</h2> <ul class="menu nav"><li class="first leaf"><a href="/search?category=Proteins%20and%20Enzymes" title="" 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IC Staining Flow Cytometry Protocol Using Detergents: R&D Systems