Immune Cell Culture: Proteins

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class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:h1_2_2_text_to_media:field_indv_h1"> <div><h1>Proteins for Immune Cell Culture</h1></div> </div> <div class="field_block:paragraph:h1_2_2_text_to_media:field_lg_sngl_frmtd_text_global"> <div><p>Optimize your immune cell cultures with R&D Systems™ cytokines. Our proteins provide superior performance and lot-to-lot consistency so you can have confidence in their ability to promote robust immune cell expansion and differentiation with minimal variability between cultures. From basic research to clinical applications, we offer research-grade, Animal-free™ RUO, and GMP-grade cytokines to meet all your experimental needs.</p></div> </div> <div class="field_block:paragraph:h1_2_2_text_to_media:field_call_to_action_global"> <a href="#key-benefits" class="btn btn-primary" type="link"> Key Benefits of R&D Systems Proteins </a> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:h1_2_2_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/banners/bt-hero-immunecell-culture-macrophage.png" alt="Immune cell" fetchpriority="high" > </div> </div> </div> </div> </div> </div> </div> </div> </div> </section> </div> </div> </header> </div> <div id="main" class="container-fluid container-wide"> <div class="row"> <div class="col-md-12"> <div> <div id="block-bio-techne-global-breadcrumbs" class="system_breadcrumb_block"> <nav role="navigation" aria-labelledby="system-breadcrumb"> <h2 id="system-breadcrumb" class="visually-hidden">Breadcrumb</h2> <ol> <li> <a href="/">Home</a> </li> <li> <a href="/reagents">Bio-Techne Reagents</a> </li> <li> <a href="/reagents/proteins">Proteins</a> </li> <li> Proteins For Immune Cell Culture </li> </ol> </nav> </div> </div> </div> </div> <main> <section class="section"> <div> <div id="block-bio-techne-global-content" class="system_main_block"> <article> <div> <div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-optimizing-your-immune-cell-cultures17" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Optimizing Your Immune Cell Cultures</h5> </a> </div> <div class="collapse" id="expPara-optimizing-your-immune-cell-cultures17" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <br/> <div><p>Immune cell culture is being increasingly utilized for investigating immune cell functions, developing assays for drug discovery, and expanding immune cell populations for cell therapies. Since there are many different types of immune cells, the culture conditions for each cell type can be highly variable. As a result, the reagents and protocols needed to maintain, expand, and differentiate different immune cells have to be tested and optimized. Once optimal culture conditions are established, researchers must ensure that these conditions are reproducible so that they can be confident that they are working with identical cultures from one experiment to the next. </p> <p>Some of the most important components for immune cell cultures are the cytokines that are used as media supplements. These cytokines are critical for maintaining healthy cells, promoting proliferation, and driving differentiation. Therefore, it is important that they exhibit high levels of activity, display minimal lot-to-lot variability, and are free of contaminants to ensure that they support cell viability and proliferation, while also contributing to the establishment of standardized, reproducible culture conditions.</p> <p>R&D Systems cytokines are designed to provide you with superior performance and consistency. Almost all of R&D Systems proteins are manufactured in-house, allowing us to maintain complete control over the quality of our products. Our rigorous in-house testing and quality control specifications ensure that our proteins will provide the highest levels of activity, low endotoxin levels (<0.1 EU/ug), and high lot-to-lot consistency. To meet your experimental needs from basic research to clinical applications, we offer different protein grades, including research-grade proteins, Animal-free Preclinical proteins, and GMP-grade proteins. Our Animal-free Preclinical and GMP-grade cytokines are manufactured using identical expression systems and purification methods to ensure a seamless transition for researchers moving from preclinical research into clinical manufacturing. With over 30 years of experience, R&D Systems has the capacity and the expertise to scale up the production of any protein, and we offer supply chain reliability so that you can be confident that the reagents you need will be available to keep your experiments on schedule.</p></div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default"> <div> <div> <div class="paragraph paragraph--type--header"> <div> <div><h2>Select the Cell Type You Are Culturing from the Immune Cell Icons Below</h2></div> </div> </div> </div> <div> <div class="paragraph paragraph--type--clickable-icons-container paragraph--view-mode--default"> <div class="container"> <div class="row"> <div class="col p-0 "> <h2></h2> </div> </div> <div class="row"> <div class="col "> </div> </div> <div class="row"> <div class="row"> <div class="col-xl-2 col-lg-2 col-md-4 col-sm-6 col-6 mb-4"> <div class="paragraph paragraph--type--indvidual-clickable-icon paragraph--view-mode--default card p-3 text-center h-100 mb-0"> <a href="#t-cells" > <div><article> <div><img src='https://resources.bio-techne.com/bio-techne-assets/images/research-areas/t-cells-icon-2.png' alt='T Cell Icon'></div> </article> </div> <p><strong>T Cells</strong></p> </a> </div> </div> <div class="col-xl-2 col-lg-2 col-md-4 col-sm-6 col-6 mb-4"> <div class="paragraph paragraph--type--indvidual-clickable-icon paragraph--view-mode--default card p-3 text-center h-100 mb-0"> <a href="#natural-killer-cells" > <div><article> <div><img src='https://resources.bio-techne.com/bio-techne-assets/images/research-areas/killer-cells-icon-2.png' alt='Killer Cell Icon'></div> </article> </div> <p><strong>Natural Killer Cells</strong></p> </a> </div> </div> <div class="col-xl-2 col-lg-2 col-md-4 col-sm-6 col-6 mb-4"> <div class="paragraph paragraph--type--indvidual-clickable-icon paragraph--view-mode--default card p-3 text-center h-100 mb-0"> <a href="#monocytes-macrophages" > <div><article> <div><img src='https://resources.bio-techne.com/bio-techne-assets/images/research-areas/monocytes-macrophages-icon-2.png' alt='Macrophage Icon'></div> </article> </div> <p><strong>Monocytes / Macrophages</strong></p> </a> </div> </div> <div class="col-xl-2 col-lg-2 col-md-4 col-sm-6 col-6 mb-4"> <div class="paragraph paragraph--type--indvidual-clickable-icon paragraph--view-mode--default card p-3 text-center h-100 mb-0"> <a href="#dendritic-cells" > <div><article> <div><img src='https://resources.bio-techne.com/bio-techne-assets/images/research-areas/dendritic-cells-icon-2.png' alt='Dendritic Cell Icon'></div> </article> </div> <p><strong>Dendritic Cells</strong></p> </a> </div> </div> <div class="col-xl-2 col-lg-2 col-md-4 col-sm-6 col-6 mb-4"> <div class="paragraph paragraph--type--indvidual-clickable-icon paragraph--view-mode--default card p-3 text-center h-100 mb-0"> <a href="#b-cells" > <div><article> <div><img src='https://resources.bio-techne.com/bio-techne-assets/images/research-areas/b-cells-icon-2.png' alt='B Cell Icon'></div> </article> </div> <p><strong>B Cells</strong></p> </a> </div> </div> <div class="col-xl-2 col-lg-2 col-md-4 col-sm-6 col-6 mb-4"> <div class="paragraph paragraph--type--indvidual-clickable-icon paragraph--view-mode--default card p-3 text-center h-100 mb-0"> <a href="#granulocytes" > <div><article> <div><img src='https://resources.bio-techne.com/bio-techne-assets/images/research-areas/granulocytes-icon-2.png' alt='Granulocyte Icon'></div> </article> </div> <p><strong>Granulocytes</strong></p> </a> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="key-benefits"></a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--header"> <div> <div><h2>Key Benefits of Using R&D Systems Cytokines for Immune Cell Culture</h2></div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><ul> <li><strong>High Levels of Biological Activity</strong>: Utilizing over 900 different bioassays, the biological activity of every protein we offer is tested in an appropriate bioassay to confirm that it meets our strict QC activity parameters.</li> <li><strong>Lot-to-Lot Consistency</strong>: Minimal lot-to-lot variability is ensured by testing each new lot side-by-side with previous lots and with a master lot, so you don’t have to worry whether results will be reproducible over time.</li> <li><strong>High Purity</strong>: Our proteins are typically over 95% pure.</li> <li><strong>Low Endotoxin Levels</strong>: Our proteins have a guaranteed industry-leading endotoxin level of <0.1 EU/ug by the LAL method.</li> <li><strong>Seamless Transition from Preclinical Research to Clinical Manufacturing</strong>: R&D Systems Animal-free RUO and Animal-free GMP-grade proteins frequently originate from the same clone, sequence, and expression system to make the transition from preclinical research into clinical manufacturing as seamless as possible.</li> <li><strong>Supply Chain Reliability</strong>: Our team has the experience and the capacity to ensure that we can provide you with a stable supply of the proteins you need for your research.</li> <li><strong>Bulk Proteins at Discounted Prices</strong>: We have the ability to scale up the production of any protein and we offer economical pricing on bulk orders.</li> <li><strong>Proteins Beyond the Catalog</strong>: We have tens of thousands of non-catalog proteins that may include different tags or come from different sources than the proteins listed on our website. <a href="https://www.rndsystems.com/services/proteins-beyond-our-catalog" target="_blank">Contact R&D Systems</a> to see if we may already have the protein that you need.</li> <li><strong>Custom Protein Capabilities</strong>: For specialized protein requests, you can always contact our <a href="/services/custom-protein-services">Custom Protein Services</a> team. We have the expertise and the systems necessary to develop the proteins required to advance your research.</li> <li><strong>Comprehensive Portfolio of Reagents for Your Entire Workflow:</strong> Along with our proteins, Bio-Techne also offers a wide range of other products for immune cell culture and characterization, including <a href="/search?keywords=%22cell%20culture%20media%22%20OR%20%22media%20supplements%22">media</a>, <a href="/search?keywords=%22cell%20culture%20media%22%20OR%20%22media%20supplements%22">media supplements</a>, <a href="/reagents/antibodies">antibodies</a>, <a href="/reagents/elisa-kits">immunoassays</a>, <a href="/reagents/rnascope-ish-technology">RNAscope™ ISH assays</a>, and <a href="/brands/proteinsimple">analytical instruments</a> to automate different steps of your workflow. </li> </ul></div> </div> </div> </div> </div> </div> <div> <section class="paragraph paragraph--type--full-width-banner-with-image mb-4 paragraph--view-mode--default paragraph-background-color--"> <div class="banner_background_image" data-bgimage="https://resources.bio-techne.com/bio-techne-assets/images/products/propak-hero-bg.jpg" style="background-repeat: no-repeat;"> <div> <div> <div> <div class="paragraph paragraph--type--_-2-text-to-media mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_text_to_media:field_header_2_global"> <div><h2>Featured Literature: IL-2 Heat Stable Agonist Protein Application Note</h2></div> </div> <div class="field_block:paragraph:2_2_text_to_media:field_lg_sngl_frmtd_text_global"> <div><p>Explore the benefits of culturing your T cells or tumor-infiltrating lymphocytes (TILs) with our new IL-2 Heat Stable Agonist Protein. Engineered using artificial intelligence (AI), this protein has enhanced stability, enabling it to withstand high temperatures and extended culture durations.</p></div> </div> <div class="field_block:paragraph:2_2_text_to_media:field_call_to_action_global"> <a href="/resources/literature/development-of-il2-heat-stable-agonist-protein-using-artificial-intelligence" class="btn btn-primary" type="link"> Download the Application Note </a> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/products/aim-homepage-img-300.png" alt="IL-2 protein ribbon structure shown on top of a computer circuit board to depict modifications of the protein by artificial intelligence" loading="lazy" > </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </section> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="t-cells"></a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default mb-4"> <div> <div> <div class="paragraph paragraph--type--_-1-text-to-media mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--75-25"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_header_2_global"> <div><h2>T Cells</h2></div> </div> <div class="field_block:paragraph:3_1_text_to_media:field_lg_sngl_frmtd_text_global"> <div><p>CD3<sup>+</sup> T cells typically make up 70-85% of the lymphocyte population in healthy human adults and 45-70% of peripheral blood mononuclear cells. They are distinguished from other lymphocytes by their expression of the T cell receptor (TCR). CD8<sup>+</sup> cytotoxic T lymphocytes (CTLs) are a subset of CD3<sup>+</sup> T cells that recognize MHC class I- bound peptide ligands and directly kill damaged, infected, and dysfunctional cells. In contrast, CD4<sup>+</sup> T cells are a subset of CD3<sup>+</sup> T cells that recognize MHC class II-bound peptide antigens and play a central role in directing adaptive immune responses.</p></div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/v2_immune-cell-culture-lp_TCells-new.png" alt="T cell" loading="lazy" > </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-isolating-and-culturing-t-cells10" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Isolating and Culturing T Cells</h5> </a> </div> <div class="collapse" id="expPara-isolating-and-culturing-t-cells10" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <br/> <div><p>CD3<sup>+</sup> T cells typically make up 70-85% of the lymphocyte population in healthy human adults and 45-70% of peripheral blood mononuclear cells (PBMCs) in healthy individuals. Following PBMC isolation from human blood by Ficoll Hypaque density centrifugation, CD3<sup>+</sup> T cells can be separated using a magnetic bead cell enrichment kit such as R&D Systems <a href="/p/cell-selection-detection-kits/magcellect-human-cd3-t-cell-isolation-kit_magh101">MagCellect<sup>TM</sup> Human CD3<sup>+</sup> T Cell Isolation Kit</a>. Using this negative selection kit, the typical recovery of CD3<sup>+</sup> T cells ranges from 45-70% and the purity of recovered CD3<sup>+</sup> T cells is typically greater than 80%.</p> <p>As the CD3<sup>+</sup> T cell population includes both CD8<sup>+</sup> cytotoxic T cells and CD4<sup>+</sup> T cells, R&D Systems <a href="/p/cell-selection-detection-kits/magcellect-human-cd8-t-cell-isolation-kit_magh112">MagCellect<sup>TM</sup> Human CD8<sup>+</sup> T Cell Isolation Kit</a>, <a href="/p/cell-selection-detection-kits/magcellect-human-naive-cd4-t-cell-isolation-kit_magh115">MagCellect Human Naïve CD4<sup>+</sup> T Cell Isolation Kit</a>, <a href="/p/cell-selection-detection-kits/magcellect-human-cd4-t-cell-isolation-kit_magh102">MagCellect Human CD4<sup>+</sup> T Cell Isolation Kit</a>, <a href="/p/cell-selection-detection-kits/magcellect-human-memory-cd4-t-cell-isolation-kit_magh116">MagCellect Human Memory CD4+ T Cell Isolation Kit</a>, or <a href="/p/cell-selection-detection-kits/magcellect-human-cd4-cd25-regulatory-t-cell-isolation-kit_magh104">MagCellect Human CD4+CD25+ Regulatory T Cell Isolation Kit</a> could also be used to isolate more specific T cell types of interest. Alternatively, T cells can be expanded from PBMCs using R&D Systems <a href="/p/cell-culture/excellerate-human-t-cell-expansion-media-xeno-free_ccm030">ExCellerate<sup>TM</sup> Human T Cell Expansion Media, Xeno-Free</a>. Using this protocol, T cells are incubated with CD3/CD28-activating beads or plate-bound antibodies and then cultured in ExCellerate media supplemented with IL-2 for 9 days.</p> <p>T cells can also be generated from pluripotent stem cells. This involves the co-culture of iPSCs from healthy donors with murine bone marrow stromal cells to generate CD34<sup>+</sup> hematopoietic progenitor cells. The hematopoietic progenitor cells are then co-cultured with DLL-overexpressing OP9 feeder cells in the presence of SCF, Flt-3 Ligand, IL-7, and IL-15 or IL-21 to promote T cell differentiation.<sup>1</sup> T cells can also be generated from iPSCs derived from peripheral blood T cells in feeder-free culture conditions.<sup>2</sup> In this protocol, embryoid bodies are generated from single cell-dissociated iPSCs and induced to differentiate into mesoderm and hematopoietic progenitor cells (HPCs) under culture conditions that include BMP-4, FGF-basic, VEGF, and the GSK-3 inhibitor, CHIR99021. HPCs are subsequently differentiated into T cells under feeder-free conditions using immobilized DLL4 and retronectin, in the presence of SCF, TPO, and Flt-3 Ligand, and IL-7.<sup>2</sup></p> <p>Following T cell isolation or expansion, naïve CD4<sup>+</sup> T helper cells can be differentiated into different T helper cell subsets based primarily upon the addition of specific cytokines to the cell culture media. The different CD4<sup>+</sup> T helper cell subsets that have been identified include T helper type 1 (Th1), Th2, Th9, Th17, and Th22 cells, along with follicular helper T (Tfh) cells, and regulatory T cells. The table below outlines the cytokines that are required for the differentiation of different Th cell subsets.</p> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td colspan="9"><strong>Cytokines Required for Naïve CD4<sup>+</sup> T Cell Differentiation into the Different T Helper Cell Subsets</strong></td> </tr> <tr> <td>Th1</td> <td>Th2</td> <td colspan="2">Th17</td> <td>Th9</td> <td>Th22</td> <td colspan="2">Tfh</td> <td>Treg</td> </tr> <tr> <td>Human/Mouse</td> <td>Human/Mouse</td> <td>Human </td> <td>Mouse</td> <td>Human/Mouse</td> <td>Human/Mouse</td> <td>Human</td> <td>Mouse</td> <td>Human/Mouse</td> </tr> <tr> <td>IFN-gamma</td> <td>IL-4</td> <td>IL-1 beta</td> <td>TGF-beta</td> <td>IL-4</td> <td>IL-6</td> <td>IL-12</td> <td>IL-6</td> <td>IL-2</td> </tr> <tr> <td>IL-12</td> <td>IL-2</td> <td>IL-6</td> <td>IL-6</td> <td>TGF-beta</td> <td>TNF-alpha</td> <td>IL-21</td> <td>IL-21</td> <td>TGF-beta</td> </tr> <tr> <td>IL-18</td> <td>IL-7</td> <td>IL-21</td> <td>IL-21</td> <td> </td> <td> </td> <td>IL-23</td> <td>ICOS Ligand</td> <td> </td> </tr> <tr> <td>IL-27</td> <td>TSLP</td> <td>IL-23</td> <td>IL-23</td> <td> </td> <td> </td> <td>ICOS Ligand</td> <td> </td> <td> </td> </tr> <tr> <td> </td> <td> </td> <td> </td> <td> </td> <td> </td> <td> </td> <td>TGF-beta</td> <td> </td> <td> </td> </tr> </tbody> </table></figure> <p>The different CD3+ T cell subsets are typically identified by flow cytometry as CD3<sup>+</sup>CD8<sup>+</sup> T cells, CD3<sup>+</sup>CD4<sup>+</sup> T cells, or CD3<sup>+</sup>CD4<sup>+</sup>CD8<sup>+</sup> double positive T cells, with different CD4<sup>+</sup> T helper cell subsets being distinguished using additional subset-specific cell surface and intracellular markers. Naïve T cells are characterized as CD45RA<sup>+</sup>, CD45RO<sup>-</sup>, CD95<sup>-</sup>, and CCR7<sup>+</sup> cells, while stem cell memory T cells (T<sub>SCM</sub>) are characterized as CD45RA<sup>+</sup>, CD45RO<sup>-</sup>, CD95<sup>+</sup>, and CCR7<sup>+</sup> cells. In contrast, effector memory T cells (T<sub>EM</sub>) are characterized as CD45RO<sup>+</sup>, CD45RA<sup>-</sup>, CD62L<sup>-</sup>, and CCR7<sup>-</sup>, and central memory T cells (T<sub>CM</sub>) are characterized as CD45RO<sup>+</sup>, CD45RA<sup>-</sup>, CD62L<sup>+</sup>, and CCR7<sup>+</sup> cells.</p> <p><strong>References</strong></p> <p>1. Zhou, Y. <em>et al</em>. (2022) <a href="https://pubmed.ncbi.nlm.nih.gov/35565395/" target="_blank">Engineering induced pluripotent stem cells for cancer immunotherapy</a>. Cancers <strong>14</strong>:2266.</p> <p>2. Iriguchi, S. <em>et al</em>. (2021) <a href="https://pubmed.ncbi.nlm.nih.gov/33462228/" target="_blank">A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy</a>. Nat. Commun. <strong>12</strong>: 430.</p></div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>Cytokines for T Cell Culture or Differentiation - Proteins by Molecule</h3> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/t/b7-h2/proteins-enzymes">B7-H2/ICOS Ligand</a></td> <td><a href="/t/bmp-4/proteins-enzymes">BMP-4</a>*</td> <td><a href="/t/fgf-basic-fgf2-bfgf/proteins-enzymes">FGF-basic</a>*</td> <td><a href="/t/flt-3-ligand-flt3l/proteins-enzymes">Flt-3 Ligand/FLT3L</a>*</td> <td><a href="/t/ifn-gamma/proteins-enzymes">IFN-γ</a>*</td> </tr> <tr> <td><a href="/t/il-1-beta-il-1f2/proteins-enzymes">IL-1β</a>*</td> <td><a href="/t/il-2/proteins-enzymes">IL-2</a>*‡</td> <td><a href="/t/il-4/proteins-enzymes">IL-4</a>*</td> <td><a href="/t/il-6/proteins-enzymes">IL-6</a>*</td> <td><a href="/t/il-7/proteins-enzymes">IL-7</a>*‡</td> </tr> <tr> <td><a href="/t/il-12/proteins-enzymes">IL-12</a></td> <td><a href="/t/il-15/proteins-enzymes">IL-15</a>*‡</td> <td><a href="/t/il-18-il-1f4/proteins-enzymes">IL-18/IL-1F4</a></td> <td><a href="/t/il-21/proteins-enzymes">IL-21</a>*</td> <td><a href="/t/il-23/proteins-enzymes">IL-23</a></td> </tr> <tr> <td><a href="/t/il-27/proteins-enzymes">IL-27</a></td> <td><a href="/t/scf-c-kit-ligand/proteins-enzymes">SCF</a>*</td> <td><a href="/t/tgf-beta-1/proteins-enzymes">TGF-β1</a>*</td> <td><a href="/t/tnf-alpha/proteins-enzymes">TNF-α</a>*</td> <td><a href="/t/thrombopoietin-tpo/proteins-enzymes">Thrombopoietin</a>*</td> </tr> </tbody> </table></figure> <p><em>*GMP-grade Proteins are available for these molecules.</em></p> <p>‡<em>Liquid formulations for GMP and animal-free preclinical grades of these cytokines are now available. <strong><a href="/reagents/proteins/liquid-cytokines-workflow">Request further information</a></strong>.</em></p> <h3>Antibodies for T Cell Activation or Differentiation - Products by Molecule</h3> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/t/cd3/antibodies">CD3</a></td> <td><a href="/t/cd28/antibodies">CD28</a></td> <td><a href="/t/ifn-gamma?category_attr=Primary%20Antibodies">IFN-γ</a></td> <td><a href="/t/il-4?category_attr=Primary%20Antibodies">IL-4</a></td> <td><a href="/t/il-12?category_attr=Primary%20Antibodies">IL-12</a></td> </tr> </tbody> </table></figure> <p> </p></div> </div> </div> <div> <div class="paragraph paragraph--type--individual-button mb-4 paragraph--view-mode--default w-100"> <div class="row"> <div class="col"> <a href="/search?keywords=cytokinegrowthfact&category_attr=Proteins%20and%20Enzymes" class="btn btn-primary float-left" type="link" > Search for Other Cytokines for T Cell Culture </a> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--_-2-media-to-text mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/t-cell-graph-new.png" alt="Expansion of human T cells cultured for 5, 7, and 9 days in ExCellerate T Cell Expansion Media supplemented with IL-2, Detection of CD3+CD8+ and CD3+CD8- T cells by flow cytometry following enrichment in ExCellerate T Cell Expansion Media" loading="lazy" > </div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_lg_sngl_frmtd_text_global"> <div><h3>Expansion of T Cells with R&D Systems RUO IL-2, Animal-Free RUO IL-2, and Animal-Free GMP-Grade IL-2 Proteins</h3> <p> </p> <p>PBMCs were isolated from 3 donors with the Fresenius Kabi Lovo® Automated Cell Processing System followed by positive selection of CD4<sup>+</sup> and CD8<sup>+</sup> T cells.<strong> </strong>Cells were expanded in a Wilson Wolf G-Rex® 6M Well Plate supplemented with 200 IU/mL of R&D Systems <a href="/p/proteins-enzymes/recombinant-human-il-2-protein-cf_bt-002">RUO Recombinant Human IL-2</a> (Catalog # BT-002), <a href="/p/proteins-enzymes/recombinant-human-il-2-protein-animal-free_bt-002-afl">Animal-free RUO Recombinant Human IL-2</a> (Catalog # BT-002-AFL), or <a href="/p/proteins-enzymes/recombinant-human-il-2-gmp-protein-cf_bt-002-gmp">Animal-free GMP-grade Recombinant Human IL-2</a> (Catalog # BT-002-GMP) for 3 days, followed by 10-11 additional days with fresh medium and the same version of IL-2. Following this time, cells were analyzed for (<strong>A</strong>) expansion (<strong>B</strong>) viability, and (<strong>C, D</strong>) phenotype. Histogram bars represent the average of 3 donors. (<strong>C, D</strong>) Cells were analyzed by flow cytometry to determine the percentages of CD4<sup>+</sup> or CD8<sup>+</sup> T stem cell memory or central memory (Tscm/Tcm) cells compared with the percentages of effector or effector memory T cells (Teff/Tem) using antibodies against CD62L and CCR7. Little difference was observed in the CD4<sup>+</sup> and CD8<sup>+</sup> T cell phenotypes generated with the different IL-2 products.</p></div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><em>*GMP-grade IL-2, IL-7, and IL-15, as well as G-Rex</em>®<em>, and Lovo</em>®<em> are available through our joint venture partnership with </em><strong><a href="/research-areas/immune-cell-therapy/scaleready" target="_self"><em>ScaleReady</em></a></strong><em>. </em><em>G-Rex</em><em>® is a registered trademark of Wilson Wolf Corporation. LOVO® is a registered trademark of Fresenius Kabi.</em></p></div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="natural-killer-cells"></a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default mb-4"> <div> <div> <div class="paragraph paragraph--type--_-1-text-to-media mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--75-25"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_header_2_global"> <div><h2>Natural Killer Cells</h2></div> </div> <div class="field_block:paragraph:3_1_text_to_media:field_lg_sngl_frmtd_text_global"> <div><p>Natural killer cells are lymphocytes belonging to the innate immune system that account for 5-20% of the lymphocyte population and up to 15% of peripheral blood mononuclear cells. They have both intrinsic cytotoxic potential and cytokine-producing capabilities and as a result, they have been suggested to be the innate counterpart of CD8<sup>+</sup> T cells. Human NK cells are phenotypically characterized as cells expressing high levels of CD56/NCAM-1 and lacking expression of CD3.</p></div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/banners/immune-cell-culture-lp_NKCells-new.png" alt="Natural Killer Cells" loading="lazy" > </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-isolating-and-culturing-natural-killer-cells7" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Isolating and Culturing Natural Killer Cells</h5> </a> </div> <div class="collapse" id="expPara-isolating-and-culturing-natural-killer-cells7" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <br/> <div><p>Natural killer cells are lymphocytes belonging to the innate immune system that account for 5-20% of the lymphocyte population in humans and up to 15% of peripheral blood mononuclear cells (PBMCs).  Following isolation of PBMCs from human blood by Ficoll Hypaque density centrifugation, natural killer cells can be separated using a magnetic bead cell enrichment kit such as R&D Systems <a href="/p/cell-selection-detection-kits/magcellect-human-nk-cell-isolation-kit_magh109">MagCellect<sup>TM</sup> Human NK Cell Isolation Kit</a>. Using this negative selection kit, the resulting cell preparation is highly enriched for natural killer cells with the typical purity of the recovered NK cells ranging from 80-90%. Alternatively, natural killer cells can be expanded from isolated PBMCs or CD3<sup>+</sup>-depleted PBMCs using R&D Systems <a href="/p/cell-culture/excellerate-human-nk-cell-expansion-media-animal-component-free_ccm037">ExCellerate™ Human NK Cell Expansion Media, Animal Component-Free</a>. NK cells are expanded by incubating PBMCs with NK cell activating beads or plate-bound antibody and then culturing the cells in the ExCellerate media supplemented with IL-2, IL-12, IL-18, and IL-21.</p> <p>Natural killer cells can also be generated from hematopoietic stem cells (HSCs) or from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) after induction of hematopoietic progenitor cells (HPCs). In this type of protocol, iPSCs are differentiated into CD34<sup>+</sup>CD45<sup>+</sup> hematopoietic progenitor cells, which are then placed on murine feeder cells in medium supplemented with IL-3, IL-7, IL-15, SCF, IL-2 and Flt-3 Ligand to promote NK cell differentiation.<sup>1, 2 </sup>Alternatively, feeder-free hESCs/hiPSCs can be used directly to form embryoid bodies in media supplemented with SCF, VEGF, BMP-4 and ROCK inhibitor followed by NK cell differentiation in media supplemented with IL-3, IL-7, IL-15, SCF, and Flt-3 Ligand.<sup>3</sup></p> <p>Human NK cells are phenotypically characterized as cells expressing high levels of the cell surface marker, CD56/NCAM-1 and lacking expression of CD3. Two different NK cell subsets have been identified in humans. The most abundant NK cell subset found in human blood has been characterized as CD3<sup>-</sup>CD56<sup>dim</sup>CD16<sup>+</sup> and is highly cytotoxic, while a second subset that is CD3<sup>-</sup>CD56<sup>bright</sup>CD16<sup>-</sup> has only weak cytotoxic potential and is primarily found in the lymph nodes.</p> <p><strong>References</strong></p> <p>1. Euchner, J. <em>et al</em>. (2021) <a href="https://pubmed.ncbi.nlm.nih.gov/34017328/" target="_blank">Natural killer cells generated from human induced pluripotent stem cells mature to CD56<sup>bright</sup>CD16<sup>+</sup>NKp80<sup>+/-</sup> <em>in vitro</em> and express KIR2DL2/DL3 and KIR3DL1</a>. Front. Immunol. <strong>12</strong>:640672.</p> <p>2. Knorr, D.A. et al. (2013) <a href="https://pubmed.ncbi.nlm.nih.gov/23515118/" target="_blank">Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy</a>. Stem Cells Transl. Med. <strong>2</strong>:274.</p> <p>3. Zhu, H. and D.S. Kaufman (2019) <a href="https://pubmed.ncbi.nlm.nih.gov/31396935/" target="_blank">An improved method to produce clinical-scale natural killer cells from human pluripotent stem cells</a>. Methods Mol. Biol. <strong>2048</strong>:107.</p></div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>Cytokines for Natural Killer (NK) Cell Culture - Proteins by Molecule</h3> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/t/bmp-4/proteins-enzymes">BMP-4</a>*</td> <td><a href="/t/flt-3-ligand-flt3l/proteins-enzymes">Flt-3 Ligand/FLT3L</a>*</td> <td><a href="/t/ifn-gamma/proteins-enzymes">IFN-γ</a>*</td> <td><a href="/t/il-2/proteins-enzymes">IL-2</a>*‡</td> <td><a href="/t/il-3/proteins-enzymes">IL-3</a>*</td> <td><a href="/t/il-7/proteins-enzymes">IL-7</a>*‡</td> </tr> <tr> <td><a href="/t/il-12/proteins-enzymes">IL-12</a></td> <td><a href="/t/il-15/proteins-enzymes">IL-15</a>*‡</td> <td><a href="/t/il-18-il-1f4/proteins-enzymes">IL-18/IL-1F4</a></td> <td><a href="/t/il-21/proteins-enzymes">IL-21</a>*</td> <td><a href="/t/scf-c-kit-ligand/proteins-enzymes">SCF</a>*</td> <td><a href="/t/vegf/proteins-enzymes">VEGF</a>*</td> </tr> </tbody> </table></figure> <p><em>*GMP-grade Proteins are available for these molecules.</em></p> <p>‡<em>Liquid formulations for GMP and animal-free preclinical grades of these cytokines are now available. <strong><a href="/reagents/proteins/liquid-cytokines-workflow">Request further information</a></strong>.</em></p></div> </div> </div> <div> <div class="paragraph paragraph--type--individual-button mb-4 paragraph--view-mode--default w-100"> <div class="row"> <div class="col"> <a href="/search?keywords=cytokinegrowthfact&category_attr=Proteins%20and%20Enzymes" class="btn btn-primary float-left" type="link" > Search for Other Cytokines for NK Cell Culture </a> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--full-page-text-with-media mb-4 paragraph--view-mode--default"> <div><h3>Expansion of Human Natural Killer Cells with ExCellerate Media and R&D Systems Cytokines</h3></div> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/immune-cell-culture-img2-525x400-4.png" alt="Expansion of human NK cells cultured in ExCellerate NK Cell Expansion Media with IL-2, IL-12, IL-18, and IL-21, Evaluation of CD3-CD56+ NK cell purity by flow cytometry following culture in ExCellerate NK Cell Expansion Media, Graph showing the purity of " loading="lazy" > </div> <div><p><strong>Expansion of Human Natural Killer Cells in ExCellerate Human NK Cell Expansion Media Supplemented with Recombinant Human IL-2, IL-12, IL-18, and IL-21. </strong>Human peripheral blood mononuclear cells (PBMCs) were expanded for 14 days in <a href="/p/cell-culture/excellerate-human-nk-cell-expansion-media-xeno-free_ccm032">ExCellerate Human NK Cell Expansion Media</a> (R&D Systems, Catalog # CCM032) using NK cell-activating beads and <a href="/p/proteins-enzymes/recombinant-human-il-2-protein_202-il">Recombinant Human IL-2</a> (R&D Systems, Catalog # 202-IL), <a href="/p/proteins-enzymes/recombinant-human-il-12-protein_219-il">Recombinant Human IL-12</a> (R&D Systems, Catalog # 219-IL), <a href="/p/proteins-enzymes/recombinant-human-il-18-il-1f4-protein_9124-il">Recombinant Human IL-18/IL-1F4</a> (R&D Systems, Catalog # 9124-IL), and <a href="/p/proteins-enzymes/recombinant-human-il-21-protein_8879-il">Recombinant Human IL-21</a> (R&D Systems, Catalog # 8879-IL). (<strong>A</strong>) NK cell expansion was evaluated at days 0, 7, 9, and 14 under these culture conditions and showed an approximately 150-fold expansion following 14 days. (<strong>B</strong>) NK cell purity was also evaluated at days 0, 7, 9, and 14 by flow cytometry using a <a href="/p/antibodies/human-cd3epsilon-fluorescein-conjugated-antibody-ucht1_fab100f">FITC-conjugated Mouse Anti-Human CD3 Monoclonal Antibody</a> (R&D Systems, Catalog # FAB100F) and an <a href="/p/antibodies/human-ncam-1-cd56-apc-conjugated-antibody-301040_fab2408a">APC-conjugated Mouse Anti-Human NCAM-1/CD56 Monoclonal Antibody</a> (R&D Systems, Catalog # FAB2408A). Flow quadrants were set based on isotype control-stained samples. Following 14 days in culture, the CD3<sup>-</sup>CD56<sup>+</sup> NK cell population showed a purity of 85.3% under these culture conditions as shown in the graph.</p></div> </div> </div> </div> </div> </div> <div> <div class="thin-banner banner_wrapper"> <div class="banner-dark"> <div> <div><h2>Bulk Protein Quantities Are Available at Discounted Prices</h2></div> <a href="/services/custom-protein-services/bulk-protein-reagents" > <button type="button" class="btn btn-primary"> Request a Bulk Quote </button> </a> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="monocytes-macrophages"></a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default mb-4"> <div> <div> <div class="paragraph paragraph--type--_-1-text-to-media mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--75-25"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_header_2_global"> <div><h2>Monocytes/Macrophages</h2></div> </div> <div class="field_block:paragraph:3_1_text_to_media:field_lg_sngl_frmtd_text_global"> <div><p>CD14<sup>+</sup> monocytes are present in large numbers in the circulation and in the periphery, typically accounting for 10-20% of peripheral blood mononuclear cells in humans. Macrophages are derived from monocytes that differentiate to become inflammatory macrophages or tissue-resident macrophages, which are involved in maintaining tissue homeostasis and carrying out specialized tissue-specific functions, depending on their tissue of residence. Monocytes and macrophages both function as professional phagocytes that ingest and process foreign materials and cell debris. They also act as antigen-presenting cells and produce cytokines, allowing them to influence adaptive immune responses.</p></div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/banners/immune-cell-culture-lp_Monocytes-Macrophages-new.png" alt="Monocytes / Macrophages" loading="lazy" > </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-isolating-and-culturing-monocytes-and-macrophages2" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Isolating and Culturing Monocytes and Macrophages</h5> </a> </div> <div class="collapse" id="expPara-isolating-and-culturing-monocytes-and-macrophages2" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <br/> <div><p>CD14<sup>+</sup> monocytes are present in large numbers in the circulation and the periphery, typically accounting for 10-20% of peripheral blood mononuclear cells (PBMCs) in humans. Following isolation of PBMCs from human blood by Ficoll Hypaque density centrifugation, CD14<sup>+</sup> monocytes can be separated using a magnetic bead cell enrichment kit such as R&D Systems <a href="/p/cell-selection-detection-kits/magcellect-human-cd14-cell-isolation-kit_magh105">MagCellect™ Human CD14<sup>+</sup> Cell Isolation Kit</a>. Using this positive selection kit, the resulting cell population is enriched for CD14<sup>+</sup> cells with a typical recovery range of 45-75% and typical purity of the recovered cells ranging from 90-97%.</p> <p>In humans, three subsets of monocytes have been characterized based on the differential expression of the cell surface markers, CD14 and CD16. Classical monocytes are the predominant subset found in human blood and have the phenotype CD14<sup>++</sup> CD16<sup>-</sup>.  The other two smaller monocyte subsets found in humans are the intermediate CD14<sup>++</sup>CD16<sup>+</sup> monocyte subset and the non-classical CD14<sup>+</sup>CD16<sup>++</sup> monocyte subset. Together, these two subsets account for less than 15% of the total monocyte population in human blood. </p> <p>Once CD14<sup>+</sup> monocytes are isolated, they can be differentiated into M1 and M2 macrophages using GM-CSF or M-CSF, respectively. Classical M1 macrophage activation is driven by microbial products and pro-inflammatory cytokines such as IFN-gamma and/or lipopolysaccharide (LPS), or TNF-alpha. M1 macrophages typically display a CD14<sup>+</sup>CD80<sup>+</sup>CD163<sup>dim</sup>CD206<sup>+</sup> phenotype and secrete pro-inflammatory factors such as TNF-alpha, IL-1 beta, and IL-12. Alternative M2 macrophage activation is driven by IL-4 and IL-13 and results in the generation of macrophages with an anti-inflammatory phenotype. M2 macrophages typically display a CD14<sup>+</sup>CD80<sup>-</sup>CD163<sup>+</sup>CD206<sup>+</sup> phenotype and secrete anti-inflammatory cytokines such as IL-10 and TGF-beta.</p> <p>Recently, researchers have also developed protocols to generate macrophages from embryonic or induced pluripotent stem cells in the presence of different combinations of cytokines.<sup>1</sup> Macrophages generated in this way have significant advantages in terms of standardizing the source and scalability, along with being able to further manipulate the cells for the development of macrophage-based cell therapies. Further research is necessary, however, to improve reprogramming methods and to determine the safety of macrophages generated from induced or embryonic stem cells in terms of biodistribution, persistence, and tumorigenicity.<sup>1</sup></p> <p><strong>References</strong></p> <p>1. Lyadova, I. and A. Vasiliev (2022) <a href="https://pubmed.ncbi.nlm.nih.gov/35725499/" target="_blank">Macrophages derived from pluripotent stem cells: prospective applications and research gaps</a>. Cell Biosci. <strong>12</strong>:96.</p></div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>Cytokines for Monocyte/Macrophage Culture - Proteins by Molecule</h3> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/t/gm-csf/proteins-enzymes">GM-CSF</a>*</td> <td><a href="/t/ifn-gamma/proteins-enzymes">IFN-γ</a>*</td> <td><a href="/t/il-3/proteins-enzymes">IL-3</a>*</td> <td><a href="/t/il-4/proteins-enzymes">IL-4</a>*</td> <td><a href="/t/il-6/proteins-enzymes">IL-6</a>*</td> </tr> <tr> <td><a href="/t/il-10/proteins-enzymes">IL-10</a>*</td> <td><a href="/t/il-13/proteins-enzymes">IL-13</a></td> <td><a href="/t/m-csf/proteins-enzymes">M-CSF</a>*</td> <td><a href="/t/scf-c-kit-ligand/proteins-enzymes">SCF</a>*</td> <td><a href="/t/thrombopoietin-tpo/proteins-enzymes">Thrombopoietin</a>*</td> </tr> </tbody> </table></figure> <p><em>*GMP-grade Proteins are available for these molecules.</em></p></div> </div> </div> <div> <div class="paragraph paragraph--type--individual-button mb-4 paragraph--view-mode--default w-100"> <div class="row"> <div class="col"> <a href="/search?keywords=cytokinegrowthfact&category_attr=Proteins%20and%20Enzymes" class="btn btn-primary float-left" type="link" > Search for Other Cytokines for Monocyte or Macrophage Cell Culture </a> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--full-page-text-with-media mb-4 paragraph--view-mode--default"> <div><h3>Differentiation and Characterization of M1 and M2 Macrophages</h3></div> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/differation-macrophages-03.png" alt="Part A: 2 by flow cytometry indicating a CD14+CD80+CD163dimCD206+ phenotype. Part B:Characterization of differentiated M2 macrophages by flow cytometry indicating a CD14+CD80-CD163+CD206+ phenotype." loading="lazy" > </div> <div><p><strong>Detection of Cell Surface Markers on M1- or M2-Activated Human Macrophages by Flow Cytometry. </strong>Enriched human CD14<sup>+</sup> monocytes were cultured in the presence of either (<strong>A</strong>) <a href="/p/proteins-enzymes/recombinant-human-gm-csf-protein_215-gm">Recombinant Human GM-CSF</a> (R&D Systems, Catalog # 215-GM) or (<strong>B</strong>) <a href="/p/proteins-enzymes/recombinant-human-m-csf-protein_216-mc">Recombinant Human M-CSF</a> (R&D Systems, Catalog # 216-MC) for 6 days in serum-free base media to promote the differentiation of M1 or M2 macrophages, respectively. On day 6 of the differentiation, cells were harvested and stained with a <a href="/p/antibodies/human-cd14-fluorescein-conjugated-antibody-134620_fab3832f">Fluorescein-conjugated Mouse Anti-Human CD14 Monoclonal Antibody</a> (open histogram; R&D Systems, Catalog # FAB3832F), a <a href="/p/antibodies/human-b7-1-cd80-pe-conjugated-antibody-37711_fab140p">PE-conjugated Mouse Anti-Human B7-1/CD80 Monoclonal Antibody</a> (open histogram; R&D Systems, Catalog # FAB140P), a <a href="/p/antibodies/human-cd163-percp-conjugated-antibody-215927_fab1607c">PerCP-conjugated Mouse Anti-Human CD163 Monoclonal Antibody</a> (open histogram; R&D Systems, Catalog # FAB1607C) and an <a href="/p/antibodies/human-mmr-cd206-apc-conjugated-antibody-685641_fab25342a">APC-conjugated Mouse Anti-Human CD206/MMR Monoclonal Antibody</a> (open histogram; R&D Systems, Catalog # FAB25342A). Cell staining was gated using isotype control antibodies (filled histograms). (<strong>A</strong>) M1 macrophages display a CD14<sup>+</sup>CD80<sup>+</sup>CD163<sup>dim</sup>CD206<sup>+</sup> phenotype while (<strong>B</strong>) M2 macrophages display a CD14<sup>+</sup>CD80<sup>-</sup>CD163<sup>+</sup>CD206<sup>+</sup>phenotype.</p></div> </div> </div> <div> <div class="paragraph paragraph--type--_-2-media-to-text mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/5ELISA-results-01-3.png" alt="Part A: ELISA results demonstrate that differentiated LPS-stimulated M1 macrophages secrete IL-12, while M2 macrophages do not. Part B: ELISA results demonstrate that differentiated LPS-stimulated M2 macrophages secrete IL-10, while M1 macrophages do not" loading="lazy" > </div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_lg_sngl_frmtd_text_global"> <div><p><strong>Differentiated Human M1 Macrophages Secrete IL-12, while Differentiated M2 Macrophages Secrete IL-10.</strong>Enriched human CD14<sup>+</sup> monocytes were cultured in the presence of either (<strong>A</strong>) <a href="/p/proteins-enzymes/recombinant-human-gm-csf-protein_215-gm">Recombinant Human GM-CSF</a> (R&D Systems, Catalog # 215-GM) or (<strong>B</strong>) <a href="/p/proteins-enzymes/recombinant-human-m-csf-protein_216-mc">Recombinant Human M-CSF</a> (R&D Systems, Catalog # 216-MC) for 6 days in serum-free base media to promote the differentiation of M1 or M2 macrophages, respectively. On day six, M1 and M2 macrophages were stimulated with 1 ug/mL LPS for 24 hours. Cell culture supernatants were collected and cytokine secretion was measured using the <a href="/p/elisa-kits/human-il-12-p70-quantikine-hs-elisa-kit_hs120">Human IL-12 p70 Quantikine™ HS ELISA Kit</a> (R&D Systems, Catalog # HS120) and the <a href="/p/elisa-kits/human-il-10-quantikine-elisa-kit_d1000b">Human IL-10 Quantikine™ ELISA Kit</a> (R&D Systems, Catalog # D1000B).</p></div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="dendritic-cells"></a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default mb-4"> <div> <div> <div class="paragraph paragraph--type--_-1-text-to-media mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--75-25"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_header_2_global"> <div><h2>Dendritic Cells</h2></div> </div> <div class="field_block:paragraph:3_1_text_to_media:field_lg_sngl_frmtd_text_global"> <div><p>Dendritic cells are a rare cell population, accounting for only 1-2% of peripheral blood mononuclear cells in healthy humans, but they are key mediators of both innate and adaptive immune responses. They upregulate MHC molecules and costimulatory receptors upon pathogen recognition, capture, process and present antigens to naïve T cells, and produce polarizing cytokines that promote pathogen-specific effector T cell differentiation and activation. Dendritic cells are a heterogeneous population in terms of locations, phenotypes, and immunological functions, which allows them to differentially shape the immune response when presented with diverse pathogens. Multiple human and mouse dendritic cell subsets have been characterized.</p></div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/banners/immune-cell-culture-lp_DendriticCells-new.png" alt="Dendritic Cells" loading="lazy" > </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-culturing-monocyte-derived-dendritic-cells13" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Culturing Monocyte-Derived Dendritic Cells</h5> </a> </div> <div class="collapse" id="expPara-culturing-monocyte-derived-dendritic-cells13" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <br/> <div><p>Monocyte-derived dendritic cells (MoDCs) are a subset of dendritic cells that are widely used in immunology research. Similar to M1 and M2 macrophages, MoDCs are also generated from CD14<sup>+</sup> monocytes. Following enrichment of CD14<sup>+</sup> monocytes with R&D Systems <a href="/p/cell-selection-detection-kits/magcellect-human-cd14-cell-isolation-kit_magh105">MagCellect™ Human CD14<sup>+</sup> Cell Isolation Kit</a>, monocytes are differentiated into immature MoDCs by culturing the cells with GM-CSF and IL-4. Immature dendritic cells are then activated with pro-inflammatory mediators, such as TNF-alpha or lipopolysaccharide (LPS), to generate mature dendritic cells. Immature dendritic cells typically express low levels of B7-1, B7-2, and MHC class II and they are negative for the cell surface marker, CD83. In contrast, mature dendritic express high levels of B7-1, B7-2, MHC class II, and CD83.</p> <p>Methods of generating dendritic cells from human embryonic stem cells (hESCs) have also been developed to increase yields, consistency, and expandability compared to MoDCs generated from PBMC-derived CD14<sup>+ </sup>monocytes.<sup>1</sup> These protocols differentiate hESCs into DCs in culture medium supplemented with BMP-4, VEGF, SCF, GM-CSF, and IL-4.<sup>1</sup> Maturation of differentiated immature DCs is achieved by culturing the ESC-derived immature DCs with TNF-alpha, IFN-gamma, PGE<sub>2</sub>, and IL-1 beta.</p> <p><strong>References</strong></p> <p>1. Silk, K.M. <em>et al</em>. (2011) <a href="https://pubmed.ncbi.nlm.nih.gov/21822895/" target="_blank">Differentiation of dendritic cells from human embryonic stem cells</a>.  Methods Mol. Biol. <strong>767</strong>:449.</p></div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>Cytokines for Dendritic Cell Culture - Proteins by Molecule</h3> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/t/bmp-4/proteins-enzymes">BMP-4</a>*</td> <td><a href="/t/flt-3-ligand-flt3l/proteins-enzymes">Flt-3 Ligand/FLT3L</a>*</td> <td><a href="/t/gm-csf/proteins-enzymes">GM-CSF</a>*</td> <td><a href="/t/ifn-gamma/proteins-enzymes">IFN-γ</a>*</td> <td><a href="/t/il-1-beta-il-1f2/proteins-enzymes">IL-1β</a>*</td> <td><a href="/t/il-3/proteins-enzymes">IL-3</a>*</td> </tr> <tr> <td><a href="/t/il-4/proteins-enzymes">IL-4</a>*</td> <td><a href="/t/m-csf/proteins-enzymes">M-CSF</a>*</td> <td><a href="/t/scf-c-kit-ligand/proteins-enzymes">SCF</a>*</td> <td><a href="/t/thrombopoietin-tpo/proteins-enzymes">Thrombopoietin</a>*</td> <td><a href="/t/tnf-alpha/proteins-enzymes">TNF-α</a>*</td> <td><a href="/t/vegf/proteins-enzymes">VEGF</a>*</td> </tr> </tbody> </table></figure> <p><em>*GMP-grade Proteins are available for these molecules.</em></p></div> </div> </div> <div> <div class="paragraph paragraph--type--individual-button mb-4 paragraph--view-mode--default w-100"> <div class="row"> <div class="col"> <a href="/search?keywords=cytokinegrowthfact&category_attr=Proteins%20and%20Enzymes" class="btn btn-primary float-left" type="link" > Search for Other Cytokines for Dendritic Cell Culture </a> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--_-2-media-to-text mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/brightfield-lps-matured-cd14-525x400-2.png" alt="Brightfield image of LPS-matured monocyte-derived dendritic cells following differentiation from CD14+ monocytes." loading="lazy" > </div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_header_2_global"> <div><h3>Differentiation and Characterization of Monocyte-Derived Dendritic Cells</h3></div> </div> <div class="field_block:paragraph:2_2_media_to_text:field_lg_sngl_frmtd_text_global"> <div><p> </p> <p><strong>Differentiation of CD14<sup>+</sup> Monocytes into LPS-matured Monocyte-derived Dendritic Cells.</strong> CD14<sup>+</sup> enriched peripheral blood mononuclear cells (PBMCs) were cultured for seven days in <a href="/p/cell-culture/stemxvivo-serum-free-dendritic-cell-base-media_ccm003">StemXVivo™ Serum-Free Dendritic Cell Base Media</a> (R&D Systems, Catalog # CCM003) supplemented with <a href="/p/proteins-enzymes/recombinant-human-il-4-protein_204-il">Recombinant Human IL-4</a> (R&D Systems, Catalog # 204-IL) and <a href="/p/proteins-enzymes/recombinant-human-gm-csf-protein_215-gm">Recombinant Human GM-CSF</a> (R&D Systems, Catalog # 215-GM) and then induced with LPS for an additional 48 hours. Representative brightfield images of the cells on day 9 are shown.</p></div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--_-2-text-to-media mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_text_to_media:field_lg_sngl_frmtd_text_global"> <div><p><strong>Phenotypic Analysis of Cultured Monocyte-derived Dendritic Cells Before and After LPS-Induced Maturation.</strong>Immature monocyte-derived dendritic cells (open histograms, dotted black line) were obtained after CD14<sup>+</sup>-enriched monocytes were cultured for seven days in <a href="/p/cell-culture/stemxvivo-serum-free-dendritic-cell-base-media_ccm003">StemXVivo Serum-Free Dendritic Cell Base Media</a> (R&D Systems, Catalog # CCM003) supplemented with <a href="/p/proteins-enzymes/recombinant-human-il-4-protein_204-il">Recombinant Human IL-4</a> (R&D Systems, Catalog # 204-IL) and <a href="/p/proteins-enzymes/recombinant-human-gm-csf-protein_215-gm">Recombinant Human GM-CSF</a> (R&D Systems, Catalog # 215-GM). Mature monocyte-derived dendritic cells (filled histograms) were obtained by culturing CD14<sup>+</sup>-enriched monocytes under the same conditions for seven days and then treating the cells with LPS for an additional 48 hours. The phenotypes of Day 7 immature monocyte-derived dendritic cells and day nine LPS-treated mature monocyte-derived dendritic cells were assessed by staining with a <a href="/p/antibodies/human-b7-1-cd80-fluorescein-conjugated-antibody-37711_fab140f">Fluorescein-conjugated Mouse Anti-Human B7-1/CD80 Monoclonal Antibody</a> (R&D Systems, Catalog # FAB140F), a <a href="/p/antibodies/human-cd83-fluorescein-conjugated-antibody-hb15e_fab1774f">Fluorescein-conjugated Mouse Anti-Human CD83 Monoclonal Antibody</a> (R&D Systems, Catalog # FAB1774F), a <a href="/p/antibodies/human-b7-2-cd86-fluorescein-conjugated-antibody-37301_fab141f">Fluorescein-conjugated Mouse Anti-Human B7-2/CD86 Monoclonal Antibody</a> (R&D Systems, Catalog # FAB141F), and an Anti-MHC Class II Antibody, or an appropriate isotype control antibody (open histograms, solid blue line), followed by flow cytometry.</p></div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/assay-graded-doses525x400-2.png" alt="Analysis of immature MoDCs and LPS-treated mature MoDCs by flow cytometry with B7-1, CD83, B7-2, and MHC antibodies " loading="lazy" > </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--_-2-text-to-media mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_text_to_media:field_lg_sngl_frmtd_text_global"> <div><p><strong>Mature Dendritic Cells Induce Proliferation of Allogeneic T Cells.</strong> CD14<sup>+</sup> monocytes were cultured for seven days in <a href="/p/cell-culture/stemxvivo-serum-free-dendritic-cell-base-media_ccm003">StemXVivo Serum-Free Dendritic Cell Base Media</a> (R&D Systems, Catalog # CCM003) supplemented with <a href="/p/proteins-enzymes/recombinant-human-il-4-protein_204-il">Recombinant Human IL-4</a> (R&D Systems, Catalog # 204-IL), <a href="/p/proteins-enzymes/recombinant-human-gm-csf-protein_215-gm">Recombinant Human GM-CSF</a> (R&D Systems, Catalog # 215-GM) and gentamycin. The cells were subsequently treated with LPS for an additional 48 hours to induce dendritic cell maturation. Graded doses of mature monocyte-derived dendritic cells were incubated with 1 x 10<sup>5</sup>autologous or allogeneic CD3<sup>+</sup> T cells for five days. <sup>3</sup>H-thymidine (<sup>3</sup>H-TdR) was added to the culture for the final 18 hours and T cell proliferation was measured using a scintillation counter. Results are presented as the mean cpm obtained from three experiments.</p></div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/allogenic-autologous-tcells525x400-3.png" alt="Assay demonstrating that graded doses of LPS-treated mature MoDCs stimulate proliferation of allogeneic CD3+ T cells " loading="lazy" > </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="thin-banner banner_wrapper"> <div class="banner-dark"> <div> <div><h2>Can’t Find the Protein that You Need?<h2></h2></h2></div> <a href="/services/custom-protein-services" > <button type="button" class="btn btn-primary"> Learn About Custom Protein Services </button> </a> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="b-cells"></a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default mb-4"> <div> <div> <div class="paragraph paragraph--type--_-1-text-to-media mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--75-25"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_header_2_global"> <div><h2>B Cells</h2></div> </div> <div class="field_block:paragraph:3_1_text_to_media:field_lg_sngl_frmtd_text_global"> <div><p>B cells represent around 5-20% of the lymphocyte population in humans and up to 15% of peripheral blood mononuclear cells. They are distinguished from other lymphocytes by their expression of the B cell receptor (BCR). B cells play a key role in the adaptive immune response as they function as antigen-presenting cells, secrete regulatory cytokines, produce antibodies, and generate immunological memory. There are multiple subsets of B cells in both humans and mice, including naïve B cells, transitional B cells, memory B cells, plasma cells, and regulatory B cells, which can be distinguished based on the expression of different markers. The cell surface marker, CD19 is the most common marker used to identify B cells, as it is stably expressed throughout B cell differentiation until the plasmablast stage.</p></div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/banners/immune-cell-culture-lp_BCells-new.png" alt="B Cells" loading="lazy" > </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-isolating-and-culturing-b-cells10" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Isolating and Culturing B Cells</h5> </a> </div> <div class="collapse" id="expPara-isolating-and-culturing-b-cells10" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <br/> <div><p>B cells represent around 5-20% of the lymphocyte population in humans and up to 15% of peripheral blood mononuclear cells (PBMCs). Following isolation of PBMCs from human blood by Ficoll Hypaque density centrifugation, B cells can be separated using a magnetic bead cell enrichment kit such as R&D Systems <a href="/p/cell-selection-detection-kits/magcellect-human-b-cell-isolation-kit_magh103">MagCellect<sup>TM</sup> Human B Cell Isolation Kit</a>. Using this negative selection kit, the typical recovery ranges from 45-65% and typical purity of the recovered B cells is greater than 80%. B cells can then further be expanded using R&D Systems <a href="/p/cell-culture/excellerate-b-cell-media-xeno-free_ccm031">ExCellerate™ Human B Cell Media, Xeno-Free</a>, supplemented with IL-4 and an anti-CD40 antibody.</p> <p>There are multiple subsets of B cells in both humans and mice, including naïve B cells, transitional B cells, memory B cells, plasma cells, and regulatory B cells, which can be distinguished based on the expression of different markers. The cell surface marker, CD19 is the most common marker used to identify B cells, as it is stably expressed throughout B cell differentiation until the plasmablast stage. In humans, naïve B cells are typically CD19<sup>+</sup>CD27<sup>-</sup>IgD<sup>+</sup> cells, while transitional B cells are characterized as CD19<sup>+</sup>CD10<sup>+/-</sup>CD24<sup>+/-</sup> CD27<sup>-</sup>IgD<sup>+</sup>. Human memory B cells and plasma cells also express CD19, and in addition, memory B cells are characterized as having high levels of CD27 and low levels of CD23/Fc epsilon RII, while plasma cells are typically characterized as cells expressing high levels of CD38 and CD138/Syndecan-1 and being IgD negative. In contrast, regulatory B cells are a unique subset of immunosuppressive B cells. These cells have been found to express high levels of the cell surface markers, CD21 and CD24, along with CD38, and they secrete IL-10 and TGF-beta.</p></div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>Cytokines for B Cell Culture - Proteins by Molecule</h3> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/t/baff-blys-tnfsf13b?category_attr=Proteins%20and%20Enzymes">BAFF/BLyS/TNFSF13B</a></td> <td><a href="/t/cd40-ligand-tnfsf5/proteins-enzymes">CD40 Ligand</a></td> <td><a href="/t/flt-3-ligand-flt3l/proteins-enzymes">Flt-3 Ligand/FLT3L</a>*</td> <td><a href="/t/ifn-gamma/proteins-enzymes">IFN-γ</a>*</td> <td><a href="/t/il-2/proteins-enzymes">IL-2</a>*‡</td> </tr> <tr> <td><a href="/t/il-4/proteins-enzymes">IL-4</a>*</td> <td><a href="/t/il-7/proteins-enzymes">IL-7</a>*‡</td> <td><a href="/t/il-10/proteins-enzymes">IL-10</a>*</td> <td><a href="/t/il-21/proteins-enzymes">IL-21</a>*</td> <td> </td> </tr> </tbody> </table></figure> <p><em>*GMP-grade Proteins are available for these molecules.</em></p> <p>‡<em>Liquid formulations for GMP and animal-free preclinical grades of this cytokine are now available. <strong><a href="/reagents/proteins/liquid-cytokines-workflow">Request further information</a></strong>.</em></p></div> </div> </div> <div> <div class="paragraph paragraph--type--individual-button mb-4 paragraph--view-mode--default w-100"> <div class="row"> <div class="col"> <a href="/search?keywords=cytokinegrowthfact&category_attr=Proteins%20and%20Enzymes" class="btn btn-primary float-left" type="link" > Search for Other Cytokines for B Cell Culture </a> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--_-2-media-to-text mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/Immune-cells-culture-img3-525x400b-2.png" alt="Graph showing robust expansion of mouse B cells in ExCellerate media with IL-4 and Anti-Mouse CD40 Antibody, Brightfield image of mouse B cell colonies expanded in ExCellerate media with IL-4 and Anti-Mouse CD40 Antibody, Flow cytometry analysis of mouse" loading="lazy" > </div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_header_2_global"> <div><h3>Expansion of Mouse B Cells with ExCellerate Media and R&D Systems Cytokines</h3></div> </div> <div class="field_block:paragraph:2_2_media_to_text:field_lg_sngl_frmtd_text_global"> <div><p> </p> <p><strong>Expansion of Mouse B Cells in ExCellerate</strong><strong> Human B Cell Expansion Media Supplemented with Recombinant Mouse IL-4 and an Anti-Mouse CD40 Antibody. </strong>Mouse B cells were isolated from splenocytes using the <a href="/p/cell-selection-detection-kits/magcellect-mouse-b-cell-isolation-kit_magm204">MagCellect™ Mouse B Cell Isolation Kit</a> (R&D Systems, Catalog # MAGM204) and expanded using <a href="/p/cell-culture/excellerate-b-cell-media-xeno-free_ccm031">ExCellerate B Cell Media</a> (R&D Systems, Catalog # CCM031) with or without <a href="/p/proteins-enzymes/recombinant-mouse-il-4-protein_404-ml">Recombinant Mouse IL-4</a> (R&D Systems, Catalog # 404-ML) and a <a href="/p/antibodies/mouse-cd40-tnfrsf5-antibody-1c10_mab440">Rat Anti-Mouse CD40/TNFRSF5 Monoclonal Antibody</a> (R&D Systems, Catalog # MAB440). (<strong>A</strong>) Expansion of mouse B cells was monitored using Rezasurin. Data show that ExCellerate B Cell Media supplemented with IL-4 and an Anti-Mouse CD40 Antibody results in robust mouse B cell expansion. (<strong>B</strong>) Representative brightfield images of mouse B cell colonies. (<strong>C</strong>) Expanded mouse B cells are B220<sup>+</sup>CD19<sup>+</sup> (>98%) and negative for both CD3 and CD4.</p></div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="granulocytes"></a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default mb-4"> <div> <div> <div class="paragraph paragraph--type--_-1-text-to-media mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--75-25"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_header_2_global"> <div><h2>Granulocytes</h2></div> </div> <div class="field_block:paragraph:3_1_text_to_media:field_lg_sngl_frmtd_text_global"> <div><p>Granulocytes are polymorphonuclear leukocytes with small cytoplasmic granules that release enzymes to defend the host against invading pathogens. Granulocytes include basophils, eosinophils, mast cells and neutrophils. All of these are terminally differentiated cells that develop in the bone marrow and function primarily as part of the innate immune response, although they may also have antigen-presenting capabilities.</p></div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:3_1_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/banners/immune-cell-culture-lp_Granulocytes-new.png" alt="Granulocytes" loading="lazy" > </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-isolating-and-culturing-granulocytes15" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Isolating and Culturing Granulocytes</h5> </a> </div> <div class="collapse" id="expPara-isolating-and-culturing-granulocytes15" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <br/> <div><p>Granulocytes are polymorphonuclear leukocytes with small cytoplasmic granules that release enzymes to defend the host against invading pathogens. Granulocytes include basophils, eosinophils, mast cells and neutrophils. Of these, neutrophils are the most abundant type of blood leukocyte, but studying them is challenging as they have a short half-life and don’t proliferate, making <em>in vitro</em> expansion impossible.<sup>1</sup> As a result, researchers have developed neutrophil cell lines or have generated neutrophils from induced pluripotent stem cells.<sup>1-3</sup> Several labs have published detailed protocols outlining how to generate functional neutrophils from iPSCs. In general, it is accomplished by co-culturing iPSCs with OP9 cells in the presence of hematopoietic cytokines such as SCF, IL-6, and G-CSF, or IL-3 and G-CSF.<sup>2-4</sup></p> <p>Eosinophils make up approximately 2-5% of the total peripheral blood leukocyte population in humans, with larger numbers being found in some tissues. They are primarily isolated from a mixed granulocytic population by negative selection using anti-CD16-conjugated magnetic beads or by fluorescence-activated cell sorting (FACS).<sup>5-7</sup> FACS sorting has also been used to study human and mouse bone marrow eosinophils following labeling with a series of eosinophil-specific markers, but a downside of this method is that the antibodies used to label the cells can result in cell activation, which limits the half-life of the cells.<sup>8</sup> In mouse, eosinophils have also been differentiated <em>in vitro</em> from mouse bone marrow progenitor cells cultured in the presence of SCF, Flt-3 ligand, and IL-5, and human eosinophils have been differentiated from progenitor cells from either the umbilical cord or the bone marrow using IL-3 and IL-5.<sup>8-12</sup> </p> <p>Mast cells undergo terminal differentiation in tissues and are therefore, found in very low numbers in the circulation. As a result, they are very difficult to isolate and instead, they are typically generated by <em>in vitro</em> differentiation of CD34<sup>+ </sup>hematopoietic progenitor cells from peripheral blood or CD133<sup>+ </sup>progenitors from cord blood.<sup>13</sup> SCF, IL-3, and IL-6 are used to drive mast cell differentiation of these progenitor cells.</p> <p>In contrast to mast cells, basophils are primarily found in the circulation rather than in tissues. They typically represent less than 1% of circulating leukocytes. Basophils are most commonly isolated from leukocyte concentrates derived from healthy blood by negative selection using immunomagnetic beads.<sup>14, 15</sup></p> <p>Cell surface and intracellular markers used to identify human or mouse basophils, eosinophils, mast cells, and neutrophils can be found using R&D Systems <a href="https://www.rndsystems.com/resources/cell-markers" target="_blank">Cell Marker Interactive Resource Tool</a> and clicking on <a href="https://www.rndsystems.com/resources/cell-markers/immune-cells/granulocytes" target="_blank">Granulocytes</a>.</p> <p><strong>References</strong></p> <p>1. Blanter, M. <em>et al</em>. (2021) <a href="https://pubmed.ncbi.nlm.nih.gov/33505167/" target="_blank">Studying neutrophil function in vitro: Cell models and environmental factors</a>. J. Inflamm. Res. <strong>14</strong>:141.</p> <p>2. Morishima, T. <em>et al</em>. (2011) <a href="https://pubmed.ncbi.nlm.nih.gov/20945397/" target="_blank">Neutrophil differentiation from human-induced pluripotent stem cells</a>. J. Cell Physiol. <strong>226</strong>:1283.</p> <p>3. Sweeney, C.L. <em>et al</em>. (2014) <a href="https://pubmed.ncbi.nlm.nih.gov/24504953/" target="_blank">Generation of functionally mature neutrophils from induced pluripotent stem cells</a>. Methods Mol Biol. <strong>1124</strong>:189.</p> <p>4. Lachmann, N. <em>et al</em>. (2015) <a href="https://pubmed.ncbi.nlm.nih.gov/25680479/" target="_blank">Large-scale hematopoietic differentiation of human induced pluripoteint stem cells provides granulocytes or macrophages for cell replacement therapies</a>. Stem Cell Rep. <strong>4</strong>:282.</p> <p>5. Hansel, T.T. <em>et al</em>. (1989) <a href="https://pubmed.ncbi.nlm.nih.gov/2547875/" target="_blank">Purification of human blood eosinophils by negative selection using immunomagnetic beads</a>v. J. Immunol. Methods <strong>122</strong>:97.</p> <p>6. Hansel. T.T. <em>et al</em>. (1991) <a href="https://pubmed.ncbi.nlm.nih.gov/1662676/" target="_blank">An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils</a>. J. Immunol. Methods <strong>145</strong>:105.</p> <p>7. Chihara, J. <em>et al</em>. (1995) <a href="https://pubmed.ncbi.nlm.nih.gov/7741183/" target="_blank">A comparative study of eosinophil isolation by different procedures of CD16-negative depletion</a>. Allergy <strong>50</strong>:11.</p> <p>8. Wong, T.W. and D.F. Jelinek (2013) <a href="https://pubmed.ncbi.nlm.nih.gov/23085531/" target="_blank">Purification of functional eosinophils from human bone marrow</a>. J. Immunol. Methods <strong>387</strong>:130.</p> <p>9. Dyer, K.D. <em>et al</em>. (2008) <a href="https://pubmed.ncbi.nlm.nih.gov/18768855/" target="_blank">Functionally competent eosinophils differentiated <em>ex vivo</em> in high purity from normal mouse bone marrow</a>. J. Immunol. <strong>181</strong>:4004.</p> <p>10. Ten, R.M. <em>et al</em>. (1991) <a href="https://pubmed.ncbi.nlm.nih.gov/1873484/" target="_blank">Eosinophil differentiation of human umbilical cord mononuclear cells and prolonged survival of mature eosinophils by murine EL-4 thymoma cell conditioned medium</a>. Cytokine<strong> 3</strong>:350.</p> <p>11. Ohashi, H. <em>et al</em>. (1999) <a href="https://pubmed.ncbi.nlm.nih.gov/9925962/" target="_blank">Effect of interleukin-3, interleukin-5, and hyaluronic acid on cultured eosinophils derived from human umbilical cord blood mononuclear cells</a>. Int. Arch. Allergy Immunol. <strong>118</strong>:44.</p> <p>12. Clutterbuck, E.J. <em>et al</em>. (1989) <a href="https://pubmed.ncbi.nlm.nih.gov/2653458/" target="_blank">Human interleukin-5 (IL-5) regulates the production of eosinophils in human bone marrow cultures: comparison and interaction with IL-1, IL-3, IL-6, and GMCSF</a>. Blood <strong>73</strong>:1504.</p> <p>13. Radinger, M. <em>et al</em>. (2010) <a href="https://pubmed.ncbi.nlm.nih.gov/20814942/" target="_blank">Generation, isolation, and maintenance of human mast cells and mast cell lines</a>. Curr. Protoc. Immunol. Ch. 7:Unit 7.37.</p> <p>14. Gibbs, B.F. <em>et al</em>. (2008) <a href="https://pubmed.ncbi.nlm.nih.gov/18201250/" target="_blank">A rapid two-step procedure for the purification of human peripheral blood basophils to near homogeneity</a>. Clin. Exp. Allergy <strong>38</strong>:480.</p> <p>15. Raap, U. <em>et al</em>. (2016) <a href="https://pubmed.ncbi.nlm.nih.gov/28000952/" target="_blank">Human basophils are a source of – and are differentially activated by – IL-31</a>. Clin. Exp. Allergy <strong>47</strong>:499.</p></div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>Cytokines for Granulocyte Culture - Proteins by Molecule</h3> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/t/g-csf/proteins-enzymes">G-CSF</a></td> <td><a href="/t/gm-csf/proteins-enzymes">GM-CSF</a>*</td> <td><a href="/t/il-3/proteins-enzymes">IL-3</a>*</td> <td><a href="/t/il-5/proteins-enzymes">IL-5</a></td> <td><a href="/t/il-6/proteins-enzymes">IL-6</a>*</td> </tr> <tr> <td><a href="/t/m-csf/proteins-enzymes">M-CSF</a>*</td> <td><a href="/t/scf-c-kit-ligand/proteins-enzymes">SCF</a>*</td> <td><a href="/t/thrombopoietin-tpo/proteins-enzymes">Thrombopoietin</a>*</td> <td><a href="/t/vegf/proteins-enzymes">VEGF</a>*</td> <td> </td> </tr> </tbody> </table></figure> <p><em>*GMP-grade Proteins are available for these molecules.</em></p></div> </div> </div> <div> <div class="paragraph paragraph--type--individual-button mb-4 paragraph--view-mode--default w-100"> <div class="row"> <div class="col"> <a href="/search?keywords=cytokinegrowthfact&category_attr=Proteins%20and%20Enzymes" class="btn btn-primary float-left" type="link" > Search for Other Cytokines for Granulocyte Culture </a> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--_-2-media-to-text mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/immune-cell-culture-lp_TPO Graph.png" alt="TPO Graph" loading="lazy" > </div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_header_2_global"> <div><h3>R&D Systems Cytokines Are Frequently More Active than Leading Competitors' Proteins</h3></div> </div> <div class="field_block:paragraph:2_2_media_to_text:field_lg_sngl_frmtd_text_global"> <div><p> </p> <p><strong>R&D Systems Recombinant Human Thrombopoietin/TPO Displays Higher Activity than Leading Competitors’ Thrombopoietin Proteins.</strong> The bioactivity of R&D Systems <a href="/p/proteins-enzymes/recombinant-human-thrombopoietin-e-coli-expressed-cf_288-tpe">Recombinant Human Thrombopoietin</a> (Catalog # 288-TPE; orange line) or recombinant human Thrombopoietin/TPO from two different competitors (blue and green lines) was assessed by measuring the ability of the proteins to stimulate proliferation of the MO7e human megakaryocytic leukemic cell line. The ED<sub>50</sub> for this effect for R&D Systems Recombinant Human Thrombopoietin is 0.05-0.5 ng/mL, which is over 2-fold more active than the two competitors’ proteins.</p></div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--_-2-media-to-text mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/immune-cell-culture-lp_TPO Graph2.png" alt="Recombinant Human SCF/c-Kit Ligand Graph" loading="lazy" > </div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_header_2_global"> <div><h3>R&D Systems Cytokines Are Rigorously Tested to Ensure Lot-to-Lot Consistency</h3></div> </div> <div class="field_block:paragraph:2_2_media_to_text:field_lg_sngl_frmtd_text_global"> <div><p> </p> <p><strong>Lot-to-Lot Consistency Testing of Recombinant Human SCF/c-kit Ligand. </strong>Two independent lots of <a href="/p/proteins-enzymes/recombinant-human-scf-protein-cf_11010-sc">Recombinant Human SCF/c-kit Ligand</a> (R&D Systems, Catalog # 11010-SC) were tested for their ability to stimulate the proliferation of TF-1 human erythroleukemic cells. The ED<sub>50</sub> for this effect is 1-5 ng/mL. Each trace on the graph represents Recombinant Human SCF/c-kit Ligand from a different manufacturing run, demonstrating the lot-to-lot consistency of the protein.</p></div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="thin-banner banner_wrapper"> <div class="banner-dark"> <div> <div><h2>Animal-Free RUO to GMP</h2></div> <a href="/gmp-products/ruo-to-gmp" > <button type="button" class="btn btn-primary"> Streamline Your Transition to GMP </button> </a> </div> </div> </div> </div> <div> <section class="paragraph paragraph--type--full-width-banner-with-image mb-4 paragraph--view-mode--default paragraph-background-color--dark"> <div class="banner_background_image" data-bgimage="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/immune-cell-culture-lp_Webinar-BG6.png" style="background-repeat: no-repeat;"> <div> <div> <div> <div class="paragraph paragraph--type--_-2-media-to-text mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_header_2_global"> <div><h2>Webinar: Key Considerations for Cytokine Supplier Selection for Cell Therapies</h2></div> </div> <div class="field_block:paragraph:2_2_media_to_text:field_call_to_action_global"> <a href="/resources/webinars/key-considerations-for-cytokine-supplier-selection" class="btn btn-primary" type="link"> Watch Cytokines for Cell Therapy Webinar </a> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </section> </div> <div> <div class="paragraph paragraph--type--card-paragraph paragraph--view-mode--default card-para-default"> <h2>Additional Immune Cell-Related Products</h2> <div class="row card_row"> <div class="col-md-6"> <div class="h-100"> <div class="bg-white border rounded flex-column d-flex h-100"> <a href="/reagents/cell-culture-reagents" > <div class="card-img-top text-center mb-3" style="background-repeat: no-repeat; background-color: #00263A; height:200px; width: 100%;"> <div class="teaser-valign-container" style=""> <div class="valign"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/banners/Animal-Free-Landing-Graphics_teaser4-4.png" alt="Cell Culture Reagents" loading="lazy" > </div> </div> </div> </a> <a class="card_title_link" href="/reagents/cell-culture-reagents" > <h3 class="card_title_htag"> <div>Cell Culture Reagents</div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Browse our complete collection of products for cell culture. From our ExCellerate™ T Cell, B Cell, or NK Cell Expansion Media and media supplements, to our CellXVivo™ Immune Cell Differentiation and Expansion Kits, we offer a comprehensive range of reagents to support immune cell expansion and differentiation.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/reagents/cell-culture-reagents" > Explore Our Cell Culture Products </a> </div> </div> </div> </div> </div> <div class="col-md-6"> <div class="h-100"> <div class="bg-white border rounded flex-column d-flex h-100"> <a href="/reagents/cell-culture-reagents/serum-free-animal-free" > <div class="teaser-card-image-crop mb-3"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/animal-free-serum-free-hero-bg-v6.jpg" alt="adherent cells supported by matrix proteins" loading="lazy" > </div> </a> <a class="card_title_link" href="/reagents/cell-culture-reagents/serum-free-animal-free" > <h3 class="card_title_htag"> <div>Serum-Free and Animal-Free Cell Culture Media</div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>The elimination of serum and animal components in culture media is an important step towards a defined cell culture process and will also improve the safety profile of your cell product and simplify regulatory pathways. We offer serum-free, animal component-free media and media supplements to enhance the consistency of your cultures.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/reagents/cell-culture-reagents/serum-free-animal-free" > Learn About Serum-Free and Animal-Free Media </a> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--card-paragraph paragraph--view-mode--default card-para-default"> <div class="row card_row"> <div class="col-md-4"> <div class="h-100"> <div class="bg-white border rounded flex-column d-flex h-100"> <a href="/applications/imaging/flow-cytometry" > <div class="teaser-card-image-crop mb-3"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/flow-cytometry-525x315.jpg" alt="Flow Cytometry eHandbook Teaser" loading="lazy" > </div> </a> <a class="card_title_link" href="/applications/imaging/flow-cytometry" > <h3 class="card_title_htag"> <div>Flow Cytometry-Validated Antibodies for Identifying Immune Cell Types</div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Bio-Techne offers an unparalleled selection of fluorochrome-conjugated R&D Systems™ and Novus Biologicals™ flow cytometry-qualified antibodies for identifying or characterizing different immune cell types in mixed population samples. 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Our analytical platforms and assays enable rapid and accurate analysis of cells, viral vectors, molecules, and contaminants.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/research-areas/cell-and-gene-therapy/analytical-solutions" > View Analytical Solutions for Cell Therapies </a> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--button-row paragraph--view-mode--default"> <div class="row"> <div class="col"> <div><h2>Resources for Studying Immune Cells</h2></div> <div class="list-grid-wrap"> <div class="row"> <div class="col-md-4"> <a href="https://www.rndsystems.com/resources/cell-markers" class="btn btn-primary" type="link"> Interactive Cell Marker Tool </a> </div> <div class="col-md-4"> <a href="https://info.bio-techne.com/flow-cytometry-handbook.html" class="btn btn-primary" type="link"> Flow Cytometry eHandbook </a> </div> <div class="col-md-4"> <a href="/resources/literature/t-cell-therapies-ebook" class="btn btn-primary" type="link"> T Cell Therapies eBook </a> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--button-row paragraph--view-mode--default"> <div class="row"> <div class="col"> <div class="list-grid-wrap"> <div class="row"> <div class="col-md-4"> <a href="/resources/literature/immune-checkpoint-targets-for-cancer-immunotherapy-research-poster" class="btn btn-primary" type="link"> Immune Checkpoint Targets Wall Poster </a> </div> <div class="col-md-4"> <a href="/resources/scientific-articles/a-simple-flow-cytometry-based-assay-for-car-detection-using-fluorokines" class="btn btn-primary" type="link"> Application Note: Using Fluorokines for CAR Detection </a> </div> <div class="col-md-4"> <a href="/resources/literature/current-and-emerging-immune-checkpoint-targets-immuno-oncology-research-ebook" class="btn btn-primary" type="link"> Current and Emerging Immune Checkpoint Targets eBook </a> </div> </div> </div> </div> </div> </div> </div> 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