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View additional product information for Dynabeads® Mouse T-Activator CD3⁄CD28⁄CD137 - FAQs (11455D)
21 product FAQs found
In nature there is a constitutive internalization and re-entry of the TCR-CD3 complex to the cell surface. When Dynabeads magnetic beads bind to CD3/CD28 on the cells in culture, the TCR-CD3 complex is internalized and then degraded. When this happens the beads do not have any target on the surface of the cells to bind to and therefore the beads will be released into the medium. The bead-cell clusters last for about 3-4 days until the beads fall off the cells.
The T-cell receptor (TCR-CD3) complex consists of several different subunits where the variable immunoglobulin domains of TCR (V) bind to the ligand. The cytoplasmic tails of the CD3 subunits and TCR interact with cytosolic signaling proteins. In this cytosolic part of the complex several types of internalization signals are located.
Constitutive internalization (without ligand binding): From the literature we understand that approximately 20 tyrosine-based internalization motifs and 2 di-leucine motifs will constitutively remove the TCR- CD3 complex from the plasma membrane without ligand binding. The recycling time is approximately 100 minutes (from plasma membrane to early endosomes and back to plasma membrane), and after 10 such cycles the TCR- CD3 complex is targeted for degradation in lysosomes.
Induced internalization (with ligand binding): The ligand-dependent internalization of the TCR-CD3 complex is a ubiquitin-dependent internalization process. Upon ligand binding, ubiquitin is coupled to a certain amino acid in the cytosolic tail of the complex, which results in a clathrin-dependent internalization of the complex followed by lysosomal degradation (the internalization reaches plateau around 2 hours (Nature 375:148 (1995)). When the beads bind to CD3-CD28, the complex will actively be internalized and degraded. When this happens the beads do not have any target on the surface of the cells to bind to and therefore the beads will be released into the medium. If the beads are not removed from the medium they can re-bind to the TCR- CD3 complex when they re-appear at the plasma membrane.
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The rule of thumb is to seed the T cells at a concentration of ~0.5-1 x 10E6 cells/mL and to split the T cells when the concentration is ~1.5-2.5 x 10E6 cells/mL.
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We recommend that you culture mouse T cells in advanced RPMI-1640 with 2 mM glutamine and 10% FBS supplemented with 2000 U/mL mouse rIL-2 (human rIL-2 could also be used). The bead-to-cell ratio is 2:1. We recommend that you start with 1-1.5 X 10E6/mL T cells in suitable culture plate or flask for expanding mouse Treg cells.
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We recommend that you culture mouse T cells in advanced RPMI-1640 with 2 mM glutamine and 10% FBS supplemented with 30 U/mL mouse rIL-2 (human rIL-2 can also be used). The optimal bead-to-target cell ratio is 1:1. For short-term activation of mouse T cells, we recommend starting with 8 x 10E4 mouse T cells/well in 100-200µL medium in 96-well culture plates. For expansion of mouse T cells, we recommend that you start with 1-1.5 x 10E6/mL T cells in suitable culture plate or flask. The bead-to-cell ratio and concentration of IL-2 is the same as for activating T cells.
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The protocol for Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Cat. No. 11131D) is optimized for the activation of naive T cells, whereas the protocol for Dynabeads Human Treg Expander (Cat. No. 11129D) is optimized for regulatory T cell expansion. Both products have the same beads and contain the same antibody clones in the same CD3:CD28 ratio.
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Yes, all T cells up-regulate CTLA-4 upon activation. However, when using Dynabeads magnetic beads for activation of T cells, no ligand for CTLA-4 (B7) is present, so T cell expansion is not inhibited. In contrast, APCs express B7 that binds CTLA-4 and cause an inhibitory signal.
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Dynabeads Human T-Activator CD3/CD28/CD137 (Cat. Nos. 11162D, 11163D) is optimized for activation of Ag-specific/primed T cells by giving a weak anti-CD3 signal and co-stimulatory signals with the anti-CD28 and anti-CD137 antibodies in close proximity. For activation and expansion of memory T cells, we recommend 1 bead per 10 target cells. For expansion of primed/antigen-specific T cells, too many beads per T cell may induce apoptosis. In contrast, the Dynabeads CD3/CD28 (without the CD137) products are optimized for activation of naive T cells which require a stronger anti-CD3 activation signal than Ag-specific/primed T cells.
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Generally we recommend re-stimulating with fresh beads between days 8-12 depending on your application. Follow the product manual for instructions on re-stimulation.
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No, it does not. It is a specific anti-CD28 antibody.
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Yes, when T cells are incubated with Dynabeads magnetic beads, they typically form visible cell-bead clusters in the wells within 12 hours. After 2-3 days the cells enter the blast stage (cell division) where they increase considerably in size and their morphology changes to a more stretched appearance. After expansion (days 8-12), they will regain their usual morphology of round and clearly defined cells.
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After 3-4 days in culture, most of the Dynabeads magnetic beads have detached from the cells.
For expansion protocols, we recommend that you remove the beads with the help of a magnet between days 8-12 depending on the assay. Pipette the bead:cell complexes thoroughly before applying to the magnet for 2 minutes, and transfer the supernatant with the released cells to a new tube. Discard the used beads and add fresh beads for re-stimulation.
For short-term activation, the cells can be lysed while they are still on the beads for molecular assays. Between days 1-3, the beads form strong clusters with the cells, thus it is hard to remove the beads in this period without losing a lot of the cells.
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No, only T cells will get the stimulation signal and grow in culture. After a week the purity of CD3+ cells is typically above 95%. Stimulating MNC can be problematic due to the presence of monocytes as they will phagocytose the beads. We recommend that you remove the monocytes from the MNC or to isolate T cells before T cell activation/expansion by standard methods such as freeze/thaw, plastic adherence, or use of Dynabeads CD14 (Cat. No. 11149D, for human RUO).
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This depends on the product you are using:
- Dynabeads Human T-Expander CD3/CD28 (Cat. No. 11141D): For T cell expansion (>3 days) use 3 beads per T cell (3:1). For re-stimulation use 1 bead per cell (1:1).
- Dynabeads Human and Mouse T Cell Activation CD3/CD28 (Cat. Nos. 11131D, 11132D, 11161D, 11456D, 11452D, 11453D): For short-termstimulation, expansion, and re-stimulation, use 1 bead per T cell (1:1).
- Dynabeads Human Treg Expander (Cat. No. 11129D): For expansion use 4 beads per T cell (4:1).
- Dynabeads Human T-Activator CD3/CD28/CD137 (Cat. No. 11162D, 11163D): For expansion and re-stimulation of T cell clones/cell lines use 1 bead per 5 - 10 T cells (1:10).
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Dynabeads CD3/CD28 is optimized for activation of naive T cells by giving a strong anti-CD3 signal and a co-stimulation signal with anti-CD28 antibody in close proximity. After stimulation, the majority of the T cells are T stem central memory/T central memory cells expressing CD62L, CD45RO, CD28, CD27, CCR7 and CD95.
Only a small fraction of the cells are effector memory T cells or terminal differentiated (TEMRA CD57+) cells, in contrast to T cells expanded with anti-CD3 antibody (OKT-3).
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CD4+ T helper cells stimulated with Dynabeads CD3/CD28 will differentiate into different T helper subsets, depending on the cytokines added during in vitro culture conditions.
- Dynabeads + IL-2 give a Th1 subset (IL-12 may also be added)
- Dynabeads + IL-4 + IL-2 give a Th2 subset
- Dynabeads +IL-1b + IL-6, IL-21 and TGFβ give a Th17 subset
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The activation of T cells happens within minutes. When you analyze the cells depends on your assay (e.g., T cell signaling: within minutes, for in vitro expansion: 8-12 days).
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Some CD8+ T cells don't express the CD28 antigen. This means that when working on CD8+ T cell clones, as opposed to CD4+ clones, it is essential to determine if the CD8+ clones are expressing CD28.
- For polyclonal expansion of CD8+, use the recommended number of beads per cell for activation and re-stimulation as described in the product manual. Do not re-stimulate before day 8-12 depending on the application (very strong cell:bead clusters are formed, and the cells are producing a lot of IL-2, which is important for growth, so the media should not be changed at this point). Another optional recommendation is to use 20-100 U IL-2 in the medium when re-stimulating, but this not critical.
- For antigen-specific/primed human CD8+ cells, we recommend using the Dynabeads Human T-Activator CD3/CD28/CD137; CD137 helps these cells to grow and expand better and Dynabeads magnetic beads coated with CD3 and CD28 antibodies only. Please see the following reference (http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3083.2011.02564.x/pdf).
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Bead size for all of the T cell activation and expansion kits is 4.5 µm.
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Dynabeads CD3/CD28 offers higher expansion rate and more consistent results than traditional methods (e.g., mitogens or soluble/plate-bound antibodies). This is due to:
- Reproducible coating of optimized antibody ratios on the beads
- The bead size is close to mammalian cells, thus truly mimicking the APC to provide three-dimensional and simultaneous signals to TCR/CD3 and CD28 on the target T cells.
- Dynabeads CD3/CD28 offer a ready-to-use format that is simple and easy to use and have 24 months shelf life from the manufacturing date.
- An expansion platform covering mouse and human research, translational research, and clinical applications
- Activates naive T cells into a more central memory phenotype (Cytotherapy 16:619 (2014)).
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In nature, activation of T cells occurs through the engagement of both the T cell receptor (TCR)/CD3 and CD28 on the T cell by the major histocompatibility complex peptide and B7 family members on the antigen-presenting cells (APC) such as dendritic cells. Both of these two stimulating signals are required for production of an effective immune response. The first signal ensures that only a T cell with a TCR specific to that peptide is activated. The only co-stimulatory receptor expressed constitutively by naive T cells is CD28, this second signal licenses the T cell to respond to an antigen. In the absence of CD28 co-stimulation, T cell receptor signaling alone results in anergy.
Dynabeads magnetic beads coated with CD3/CD28 antibodies are artificial APCs (aAPC) and mimic the natural activation of T cells.
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Here are a few tips to keep the optimal cell culture growth conditions:
- It is important to keep the cell density at 0.5-1.5 x 10E6 cells/mL for optimal growth. When the cell density exceeds 1.5-2.5 x 10E6 cells/mL, re-stimulation is required.
- Re-stimulate every 8-12 days with bead:cell ratio according to the product manual.
- Activate with the correct bead:cell ratio for each specific product according to the manual. Too many beads can inhibit cell activation/proliferation.
- Use optimized cell culture media, add:
-- Growth factors (IL-2, IL-7, IL-15, or other cytokines)
-- Growth enhancing supplements (e.g., human serum/FBS, L-glutamine/glutamax, radical scavenger (10 mM N-acetyl cysteine
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