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.
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.
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 RUO proteins, and GMP-grade proteins. Our Animal-free RUO 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.
Key Benefits of Using R&D Systems Cytokines for Immune Cell Culture
- High Levels of Biological Activity: 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.
- Lot-to-Lot Consistency: 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.
- High Purity: Our proteins are typically over 95% pure.
- Low Endotoxin Levels: Our proteins have a guaranteed industry-leading endotoxin level of <0.1 EU/ug by the LAL method.
- Seamless Transition from Preclinical Research to Clinical Manufacturing: 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.
- Supply Chain Reliability: 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.
- Bulk Proteins at Discounted Prices: We have the ability to scale up the production of any protein and we offer economical pricing on bulk orders.
- Proteins Beyond the Catalog: 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. Contact R&D Systems to see if we may already have the protein that you need.
- Custom Protein Capabilities: For specialized protein requests, you can always contact our Custom Protein Services team. We have the expertise and the systems necessary to develop the proteins required to advance your research.
- Comprehensive Portfolio of Reagents for Your Entire Workflow: Along with our proteins, Bio-Techne also offers a wide range of other products for immune cell culture and characterization, including media, media supplements, antibodies, immunoassays, RNAscope™ ISH assays, and analytical instruments to automate different steps of your workflow.
CD3+ 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+ cytotoxic T lymphocytes (CTLs) are a subset of CD3+ T cells that recognize MHC class I- bound peptide ligands and directly kill damaged, infected, and dysfunctional cells. In contrast, CD4+ T cells are a subset of CD3+ T cells that recognize MHC class II-bound peptide antigens and play a central role in directing adaptive immune responses.
CD3+ 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+ T cells can be separated using a magnetic bead cell enrichment kit such as R&D Systems MagCellectTM Human CD3+ T Cell Isolation Kit. Using this negative selection kit, the typical recovery of CD3+ T cells ranges from 45-70% and the purity of recovered CD3+ T cells is typically greater than 80%.
As the CD3+ T cell population includes both CD8+ cytotoxic T cells and CD4+ T cells, R&D Systems MagCellectTM Human CD8+ T Cell Isolation Kit, MagCellect Human Naïve CD4+ T Cell Isolation Kit, MagCellect Human CD4+ T Cell Isolation Kit, MagCellect Human Memory CD4+ T Cell Isolation Kit, or MagCellect Human CD4+CD25+ Regulatory T Cell Isolation Kit 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 ExCellerateTM Human T Cell Expansion Media, Xeno-Free. 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.
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+ 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.1 T cells can also be generated from iPSCs derived from peripheral blood T cells in feeder-free culture conditions.2 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-7.2
Following T cell isolation or expansion, naïve CD4+ 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+ 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.
|Cytokines Required for Naïve CD4+ T Cell Differentiation into the Different T Helper Cell Subsets|
The different CD3+ T cell subsets are typically identified by flow cytometry as CD3+CD8+ T cells, CD3+CD4+ T cells, or CD3+CD4+CD8+ double positive T cells, with different CD4+ T helper cell subsets being distinguished using additional subset-specific cell surface and intracellular markers. Naïve T cells are characterized as CD45RA+, CD45RO-, CD95-, and CCR7+ cells, while stem cell memory T cells (TSCM) are characterized as CD45RA+, CD45RO-, CD95+, and CCR7+ cells. In contrast, effector memory T cells (TEM) are characterized as CD45RO+, CD45RA-, CD62L-, and CCR7-, and central memory T cells (TCM) are characterized as CD45RO+, CD45RA-, CD62L+, and CCR7+ cells.
1. Zhou, Y. et al. (2022) Engineering induced pluripotent stem cells for cancer immunotherapy. Cancers 14:2266.
2. Iriguchi, S. et al. (2021) A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. Nat. Commun. 12: 430.
Cytokines for T Cell Culture or Differentiation - Proteins by Molecule
|B7-H2/ICOS Ligand||BMP-4*||FGF-basic*||Flt-3 Ligand/FLT3L*||IFN-γ*|
*GMP-grade Proteins are available for these molecules.
Antibodies for T Cell Activation or Differentiation - Products by Molecule
Expansion of T Cells with R&D Systems RUO IL-2, Animal-Free RUO IL-2, and Animal-Free GMP-Grade IL-2 Proteins
PBMCs were isolated from 3 donors with the Fresenius Kabi Lovo® Automated Cell Processing System followed by positive selection of CD4+ and CD8+ T cells. Cells were expanded in a Wilson Wolf G-Rex® 6M Well Plate supplemented with 200 IU/mL of R&D Systems RUO Recombinant Human IL-2 (Catalog # BT-002), Animal-free RUO Recombinant Human IL-2 (Catalog # BT-002-AFL), or Animal-free GMP-grade Recombinant Human IL-2 (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 (A) expansion (B) viability, and (C, D) phenotype. Histogram bars represent the average of 3 donors. (C, D) Cells were analyzed by flow cytometry to determine the percentages of CD4+ or CD8+ 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+ and CD8+ T cell phenotypes generated with the different IL-2 products.
*GMP-grade IL-2, IL-7, and IL-15, as well as G-Rex®, and Lovo® are available through our joint venture partnership with ScaleReady. G-Rex® is a registered trademark of Wilson Wolf Corporation. LOVO® is a registered trademark of Fresenius Kabi.
Natural Killer Cells
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+ T cells. Human NK cells are phenotypically characterized as cells expressing high levels of CD56/NCAM-1 and lacking expression of CD3.
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 MagCellectTM Human NK Cell Isolation Kit. 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+-depleted PBMCs using R&D Systems ExCellerate™ Human NK Cell Expansion Media, Xeno-Free. 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.
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+CD45+ 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.1, 2 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.3
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-CD56dimCD16+ and is highly cytotoxic, while a second subset that is CD3-CD56brightCD16- has only weak cytotoxic potential and is primarily found in the lymph nodes.
1. Euchner, J. et al. (2021) Natural killer cells generated from human induced pluripotent stem cells mature to CD56brightCD16+NKp80+/-in vitro and express KIR2DL2/DL3 and KIR3DL1. Front. Immunol. 12:640672.
2. Knorr, D.A. et al. (2013) Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy. Stem Cells Transl. Med. 2:274.
3. Zhu, H. and D.S. Kaufman (2019) An improved method to produce clinical-scale natural killer cells from human pluripotent stem cells. Methods Mol. Biol. 2048:107.
Expansion of Human Natural Killer Cells with ExCellerate Media and R&D Systems Cytokines
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. Human peripheral blood mononuclear cells (PBMCs) were expanded for 14 days in ExCellerate Human NK Cell Expansion Media (R&D Systems, Catalog # CCM032) using NK cell-activating beads and Recombinant Human IL-2 (R&D Systems, Catalog # 202-IL), Recombinant Human IL-12 (R&D Systems, Catalog # 219-IL), Recombinant Human IL-18/IL-1F4 (R&D Systems, Catalog # 9124-IL), and Recombinant Human IL-21 (R&D Systems, Catalog # 8879-IL). (A) 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. (B) NK cell purity was also evaluated at days 0, 7, 9, and 14 by flow cytometry using a FITC-conjugated Mouse Anti-Human CD3 Monoclonal Antibody (R&D Systems, Catalog # FAB100F) and an APC-conjugated Mouse Anti-Human NCAM-1/CD56 Monoclonal Antibody (R&D Systems, Catalog # FAB2408A). Flow quadrants were set based on isotype control-stained samples. Following 14 days in culture, the CD3-CD56+ NK cell population showed a purity of 85.3% under these culture conditions as shown in the graph.
CD14+ 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.
CD14+ 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+ monocytes can be separated using a magnetic bead cell enrichment kit such as R&D Systems MagCellect™ Human CD14+ Cell Isolation Kit. Using this positive selection kit, the resulting cell population is enriched for CD14+ cells with a typical recovery range of 45-75% and typical purity of the recovered cells ranging from 90-97%.
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++ CD16-. The other two smaller monocyte subsets found in humans are the intermediate CD14++CD16+ monocyte subset and the non-classical CD14+CD16++ monocyte subset. Together, these two subsets account for less than 15% of the total monocyte population in human blood.
Once CD14+ 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+CD80+CD163dimCD206+ 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+CD80-CD163+CD206+ phenotype and secrete anti-inflammatory cytokines such as IL-10 and TGF-beta.
Recently, researchers have also developed protocols to generate macrophages from embryonic or induced pluripotent stem cells in the presence of different combinations of cytokines.1 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.1
1. Lyadova, I. and A. Vasiliev (2022) Macrophages derived from pluripotent stem cells: prospective applications and research gaps. Cell Biosci. 12:96.
Differentiation and Characterization of M1 and M2 Macrophages
Detection of Cell Surface Markers on M1- or M2-Activated Human Macrophages by Flow Cytometry. Enriched human CD14+ monocytes were cultured in the presence of either (A) Recombinant Human GM-CSF (R&D Systems, Catalog # 215-GM) or (B) Recombinant Human M-CSF (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 Fluorescein-conjugated Mouse Anti-Human CD14 Monoclonal Antibody (open histogram; R&D Systems, Catalog # FAB3832F), a PE-conjugated Mouse Anti-Human B7-1/CD80 Monoclonal Antibody (open histogram; R&D Systems, Catalog # FAB140P), a PerCP-conjugated Mouse Anti-Human CD163 Monoclonal Antibody (open histogram; R&D Systems, Catalog # FAB1607C) and an APC-conjugated Mouse Anti-Human CD206/MMR Monoclonal Antibody (open histogram; R&D Systems, Catalog # FAB25342A). Cell staining was gated using isotype control antibodies (filled histograms). (A) M1 macrophages display a CD14+CD80+CD163dimCD206+ phenotype while (B) M2 macrophages display a CD14+CD80-CD163+CD206+phenotype.
Differentiated Human M1 Macrophages Secrete IL-12, while Differentiated M2 Macrophages Secrete IL-10.Enriched human CD14+ monocytes were cultured in the presence of either (A) Recombinant Human GM-CSF (R&D Systems, Catalog # 215-GM) or (B) Recombinant Human M-CSF (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 Human IL-12 p70 Quantikine™ HS ELISA Kit (R&D Systems, Catalog # HS120) and the Human IL-10 Quantikine™ ELISA Kit (R&D Systems, Catalog # D1000B).
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.
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+ monocytes. Following enrichment of CD14+ monocytes with R&D Systems MagCellect™ Human CD14+ Cell Isolation Kit, 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.
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+ monocytes.1 These protocols differentiate hESCs into DCs in culture medium supplemented with BMP-4, VEGF, SCF, GM-CSF, and IL-4.1 Maturation of differentiated immature DCs is achieved by culturing the ESC-derived immature DCs with TNF-alpha, IFN-gamma, PGE2, and IL-1 beta.
1. Silk, K.M. et al. (2011) Differentiation of dendritic cells from human embryonic stem cells. Methods Mol. Biol. 767:449.
Differentiation and Characterization of Monocyte-Derived Dendritic Cells
Differentiation of CD14+ Monocytes into LPS-matured Monocyte-derived Dendritic Cells. CD14+ enriched peripheral blood mononuclear cells (PBMCs) were cultured for seven days in StemXVivo™ Serum-Free Dendritic Cell Base Media (R&D Systems, Catalog # CCM003) supplemented with Recombinant Human IL-4 (R&D Systems, Catalog # 204-IL) and Recombinant Human GM-CSF (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.
Phenotypic Analysis of Cultured Monocyte-derived Dendritic Cells Before and After LPS-Induced Maturation.Immature monocyte-derived dendritic cells (open histograms, dotted black line) were obtained after CD14+-enriched monocytes were cultured for seven days in StemXVivo Serum-Free Dendritic Cell Base Media (R&D Systems, Catalog # CCM003) supplemented with Recombinant Human IL-4 (R&D Systems, Catalog # 204-IL) and Recombinant Human GM-CSF (R&D Systems, Catalog # 215-GM). Mature monocyte-derived dendritic cells (filled histograms) were obtained by culturing CD14+-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 Fluorescein-conjugated Mouse Anti-Human B7-1/CD80 Monoclonal Antibody (R&D Systems, Catalog # FAB140F), a Fluorescein-conjugated Mouse Anti-Human CD83 Monoclonal Antibody (R&D Systems, Catalog # FAB1774F), a Fluorescein-conjugated Mouse Anti-Human B7-2/CD86 Monoclonal Antibody (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.
Mature Dendritic Cells Induce Proliferation of Allogeneic T Cells. CD14+ monocytes were cultured for seven days in StemXVivo Serum-Free Dendritic Cell Base Media (R&D Systems, Catalog # CCM003) supplemented with Recombinant Human IL-4 (R&D Systems, Catalog # 204-IL), Recombinant Human GM-CSF (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 105autologous or allogeneic CD3+ T cells for five days. 3H-thymidine (3H-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.
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.
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 MagCellectTM Human B Cell Isolation Kit. 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 ExCellerate™ Human B Cell Media, Xeno-Free, supplemented with IL-4 and an anti-CD40 antibody.
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+CD27-IgD+ cells, while transitional B cells are characterized as CD19+CD10+/-CD24+/- CD27-IgD+. 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.
Expansion of Mouse B Cells with ExCellerate Media and R&D Systems Cytokines
Expansion of Mouse B Cells in ExCellerate Human B Cell Expansion Media Supplemented with Recombinant Mouse IL-4 and an Anti-Mouse CD40 Antibody. Mouse B cells were isolated from splenocytes using the MagCellect™ Mouse B Cell Isolation Kit (R&D Systems, Catalog # MAGM204) and expanded using ExCellerate B Cell Media (R&D Systems, Catalog # CCM031) with or without Recombinant Mouse IL-4 (R&D Systems, Catalog # 404-ML) and a Rat Anti-Mouse CD40/TNFRSF5 Monoclonal Antibody (R&D Systems, Catalog # MAB440). (A) 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. (B) Representative brightfield images of mouse B cell colonies. (C) Expanded mouse B cells are B220+CD19+ (>98%) and negative for both CD3 and CD4.
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.
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 in vitro expansion impossible.1 As a result, researchers have developed neutrophil cell lines or have generated neutrophils from induced pluripotent stem cells.1-3 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.2-4
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).5-7 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.8 In mouse, eosinophils have also been differentiated in vitro 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-5.8-12
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 in vitro differentiation of CD34+ hematopoietic progenitor cells from peripheral blood or CD133+ progenitors from cord blood.13 SCF, IL-3, and IL-6 are used to drive mast cell differentiation of these progenitor cells.
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.14, 15
Cell surface and intracellular markers used to identify human or mouse basophils, eosinophils, mast cells, and neutrophils can be found using R&D Systems Cell Marker Interactive Resource Tool and clicking on Granulocytes.
1. Blanter, M. et al. (2021) Studying neutrophil function in vitro: Cell models and environmental factors. J. Inflamm. Res. 14:141.
2. Morishima, T. et al. (2011) Neutrophil differentiation from human-induced pluripotent stem cells. J. Cell Physiol. 226:1283.
3. Sweeney, C.L. et al. (2014) Generation of functionally mature neutrophils from induced pluripotent stem cells. Methods Mol Biol. 1124:189.
4. Lachmann, N. et al. (2015) Large-scale hematopoietic differentiation of human induced pluripoteint stem cells provides granulocytes or macrophages for cell replacement therapies. Stem Cell Rep. 4:282.
5. Hansel, T.T. et al. (1989) Purification of human blood eosinophils by negative selection using immunomagnetic beadsv. J. Immunol. Methods 122:97.
6. Hansel. T.T. et al. (1991) An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Immunol. Methods 145:105.
7. Chihara, J. et al. (1995) A comparative study of eosinophil isolation by different procedures of CD16-negative depletion. Allergy 50:11.
8. Wong, T.W. and D.F. Jelinek (2013) Purification of functional eosinophils from human bone marrow. J. Immunol. Methods 387:130.
9. Dyer, K.D. et al. (2008) Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow. J. Immunol. 181:4004.
10. Ten, R.M. et al. (1991) Eosinophil differentiation of human umbilical cord mononuclear cells and prolonged survival of mature eosinophils by murine EL-4 thymoma cell conditioned medium. Cytokine 3:350.
11. Ohashi, H. et al. (1999) Effect of interleukin-3, interleukin-5, and hyaluronic acid on cultured eosinophils derived from human umbilical cord blood mononuclear cells. Int. Arch. Allergy Immunol. 118:44.
12. Clutterbuck, E.J. et al. (1989) 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. Blood 73:1504.
13. Radinger, M. et al. (2010) Generation, isolation, and maintenance of human mast cells and mast cell lines. Curr. Protoc. Immunol. Ch. 7:Unit 7.37.
14. Gibbs, B.F. et al. (2008) A rapid two-step procedure for the purification of human peripheral blood basophils to near homogeneity. Clin. Exp. Allergy 38:480.
15. Raap, U. et al. (2016) Human basophils are a source of – and are differentially activated by – IL-31. Clin. Exp. Allergy 47:499.
R&D Systems Cytokines Are Frequently More Active than Leading Competitors' Proteins
R&D Systems Recombinant Human Thrombopoietin/TPO Displays Higher Activity than Leading Competitors’ Thrombopoietin Proteins. The bioactivity of R&D Systems Recombinant Human Thrombopoietin (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 ED50 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.
R&D Systems Cytokines Are Rigorously Tested to Ensure Lot-to-Lot Consistency
Lot-to-Lot Consistency Testing of Recombinant Human SCF/c-kit Ligand. Two independent lots of Recombinant Human SCF/c-kit Ligand (R&D Systems, Catalog # 11010-SC) were tested for their ability to stimulate the proliferation of TF-1 human erythroleukemic cells. The ED50 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.
Additional Immune Cell-Related Products
Cell Culture Reagents
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 FBS and CellXVivo™ Immune Cell Differentiation and Expansion Kits, we offer a comprehensive range of reagents to support immune cell expansion and differentiation.
Serum-Free and Animal-Free Cell Culture Media
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.
Flow Cytometry-Validated Antibodies for Identifying Immune Cell Types
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. Choose from an expansive collection of both proprietary antibodies and some of the most highly referenced antibody clones on the market.
Through the Tocris brand, Bio-techne offers a range of ready-to-conjugate dyes, from traditional small organic dyes to exclusive new technology, providing scientists with a full spectrum of choices for biomolecule conjugation and application.
Immunoassays for Cytokine Detection
From our complete, ready-to-use Quantikine™ ELISA Kits to our multiplex Luminex® Assays and fully automated Simple Plex™ Assays, you can count on our immunoassays to deliver accurate, reproducible, high-quality data for every experimental sample that you test.
Immune Cell Therapy-Related Products
Preclinical Animal-Free™ RUO and GMP-Grade Recombinant Proteins
Bio-Techne offers both R&D Systems Animal-free RUO Proteins and Animal-free GMP-grade Proteins, which are manufactured using identical expression systems and purification methods to ensure a seamless transition for researchers moving from preclinical research into clinical manufacturing.
Fluorokines™ Fluorescent-Labeled Proteins
Utilize our wide selection of fluorescent-labeled recombinant proteins to evaluate the expression of specific chimeric antigen receptors (CARs) on CAR-T or CAR-NK cells. Fluorokines are highly specific and allow CAR-expressing cells to be directly stained and detected by flow cytometry.
Organoid and 3-D Cell Culture Products
Co-culture of immune cells with tumor organoids is being increasingly utilized to study interactions that may occur in tumor microenvironments and to evaluate potential immunotherapies. Bio-Techne offers a complete collection of organoid culture reagents that are designed to promote robust organoid growth, while maintaining consistent, reproducible culture conditions.
Analytical Solutions for Cell Therapies
Rigorously characterize your mid-process and final cell therapy products to ensure the safest possible therapies. Our analytical platforms and assays enable rapid and accurate analysis of cells, viral vectors, molecules, and contaminants.