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View additional product information for Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation - FAQs (11131D, 11132D, 11161D)
50 product FAQs found
Please review the following possibilities for why your Dynabeads magnetic beads are not pelleting:
- The solution is too viscous.
- The beads have formed aggregates because of protein-protein interaction.
Try these suggestions:
- Increase separation time (leave tub on magnet for 2-5 minutes)
- Add DNase I to the lysate (~0.01 mg/mL)
- Increase the Tween 20 concentration to ~0.05% of the binding and/or washing buffer.
- Add up to 20 mM beta-merecaptoethanol to the binding and/or wash buffers.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
For biotin-labeled DNA that is less than 1 kb, we recommend you use Dynabeads M270 Streptavidin (Cat. No. 65305) and MyOne C1 magnetic beads (Cat. No. 65001). We recommend our Dynabeads KilobaseBINDER Kit (Cat. No. 60101), which is designed to immobilize long (>1 kb) double-stranded DNA molecules. The KilobaseBINDER reagent consists of M-280 Streptavidin-coupled Dynabeads magnetic beads along with a patented immobilization activator in the binding solution to bind to long, biotinylated DNA molecules for isolation. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/immobilisation-of-long-biotinylated-dna-fragments.html) for more information in regards to long biotinylated DNA fragment isolation.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Yes, Dynabeads magnetic beads can be used to isolate single-stranded DNA. Streptavidin Dynabeads magnetic beads can be used to target biotinylated DNA fragments, followed by denaturation of the double-stranded DNA and removal of the non-biotinylated strand. The streptavidin-coupled Dynabeads magnetic beads will not inhibit any enzymatic activity. This enables further handling and manipulation of the bead-bound DNA directly on the solid phase. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/preparing-single-stranded-dna-templates.html) for more information in regards to single-stranded DNA capture.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Magnetic susceptibility is a measure of how quickly the beads will migrate to the magnet. This will depend on the iron content and the character of the iron oxide. The magnetic susceptibility given for the Dynabeads magnetic beads is the mass susceptibility, given either as cgs units/g or m^3/kg (the latter being an SI unit). For ferri- and ferromagnetic substances, the magnetic mass susceptibility is dependent upon the magnetic field strength (H), as the magnetization of such substances is not a linear function of H but approaches a saturation value with increasing field. For that reason, the magnetic mass susceptibility of the Dynabeads magnetic beads is determined by a standardized procedure under fixed conditions. The magnetic mass susceptibility given in our catalog is thus the SI unit. Conversion from Gaussian (cgs, emu) units into SI units for magnetic mass susceptibility is achieved by multiplying the Gaussian factor (emu/g or cgs/g) by 4 pi x 10^-3. The resulting unit is also called the rationalized magnetic mass susceptibility, which should be distinguished from the (SI) dimensionless magnetic susceptibility unit. In general, magnetic mass susceptibility is a measure of the force (Fz) influencing an object positioned in a nonhomogenous magnetic field. The magnetic mass susceptibility of the Dynabeads magnetic beads is measured by weighing a sample, and then subjecting the sample to a magnetic field of known strength. The weight (F1) is then measured, and compared to the weight of the sample when the magnetic field is turned off (F0). The susceptibility is then calculated as K x 10^-3 = [(F1-F0) x m x 0.335 x 10^6], where K is the mass susceptibility of the sample of mass m. The susceptibility is then converted to SI units.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
There are different methods to check binding of ligands to the beads, including optical density (OD) measurement, fluorescent labeling, and radioactive labeling.
For OD measurement, you would measure the OD of the ligand before immobilization to the beads and compare it with the ligand concentration that is left in the supernatant after coating. This gives a crude measurement of how much protein has bound to the beads.
Protocol:
1.Set spectrophotometer to the right wavelength. As a blank, use the Coupling Buffer.
2.Measure the absorbance of the Pre-Coupling Solution. A further dilution may be necessary to read the absorbance, depending upon the amount of ligand added.
3.Measure the absorbance of the Post-Coupling Solution. A dilution may be necessary to read the absorbance.
4.Calculate the coupling efficiency, expressed as the % protein uptake, as follows. [(Pre-Coupling Solution x D) - (Post-Coupling Solution x D)] x 100/(Pre-Coupling Solution x D) where D = dilution factor.
For fluorescent labeling, we suggest negatively quantifying the amount of ligand bound by measuring ligand remaining in the coupling supernatant (compared to the original sample), rather than directly measuring the ligands on the beads. Add labeled ligand to the beads, and measure how much ligand is left in the supernatant (not bound to the beads). By comparing this with the total amount added in the first place, you can then calculate how much of the ligand that has been bound to the beads. Keep in mind that the Dynabeads magnetic beads are also autofluorescent, which is why direct measuring of fluorescence of the bead-bound ligands is not recommended, but rather this indirect approach. The label could be, for example, FITC/PE. Some researchers perform a direct approach with success (using a flow cytometer).
Radioactive labeling is the most sensitive method of the three, but it is also the most difficult one. It involves radioactively labeling a portion of the ligand. We use radiolabeled I-125 in tracer amounts and mix it with "cold" ligands in a known ratio before coupling. The absolute quantities for the ligand on the beads should be obtained by measuring the beads in a scintillation (gamma) counter and comparing the cpm with a standard.
Protocol:
1.Take out an appropriate amount of beads and wash the beads in 1 mL of binding buffer.
2.Pipette out desired amount of human IgG in a separate tube.
3.Mix the human IgG with I-125-labeled human IgG (30,000 - 100,000 cpm).
4.Dilute the mixture of human IgG and I-125-labeled human IgG to 100 mL in binding buffer.
5.Incubate for 30 minutes at room temperature and measure the cpm in a scintillation counter.
6.Wash the beads (with coating) four times, and measure cpm again.
The % binding is calculated by using the equation : (cpm after washing/cpm before washing)x100%.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Dynabeads magnetic beads come in three sizes: 4.5 µm (M-450), 2.8 µm (M-270/M-280), and 1 µm (MyOne beads). The largest of the Dynabeads magnetic beads is ideal for big targets like cells. The 2.8 µm beads are recommended for proteomics and molecular applications. The smallest of the beads, 1 µm, are ideal for automated handling.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
In general, short sonication is a good way to reduce aggregation of the beads and ensure optimal homogenous conditions at the time of ligand addition when coating the beads. When target is bound to the beads, more care is needed, as the binding might break. The streptavidin beads themselves should tolerate sonication. We have not tested sonication for long periods, but 5 minutes is fine. We do not have information about the streptavidin-biotin interaction being broken by such treatment.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
If desired, the uncoated epoxy or tosylactivated beads can be sterilized by washing with 70% ethanol. Coated beads cannot be sterilized.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
Dynabeads magnetic beads are uniform, non-porous, superparamagnetic, monodispersed and highly cross-linked polystyrene microspheres consisting of an even dispersion of magnetic material throughout the bead. The magnetic material within the Dynabeads magnetic beads consists of a mixture of maghemite (gamma-Fe2O3) and magnetite (Fe3O4). The iron content (Fe) of the beads is 12% by weight in Dynabeads magnetic beads M-280 and 20% by weight in Dynabeads magnetic beads M-450. The Dynabeads magnetic beads are coated with a thin polystyrene shell which encases the magnetic material, and prevents any leakage from the beads or trapping of ligands in the bead interior. The shell also protects the target from exposure to iron while providing a defined surface area for the adsorption or coupling of various molecules.
Uniformity of bead size and shape provides consistent physical and chemical properties. These uniform physical characteristics lead to high-quality, reproducible results.
The Dynabeads magnetic beads are available in three different sizes: 4.5 µm (M-450 beads), 2.8 µm (M-270/M-280 beads) and 1 µm (MyOne beads).
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
Both bead-to-target cell ratio and the concentration of beads in the bead/cell mixture are important and should be considered. For example, when using the Dynabeads magnetic beads M-450 CD4 positive isolation or depletion kit, a 4:1 bead-to-target cell ratio should be maintained. To capture 95% of target cells for molecular applications, the bead concentration must always be 1 x 10e7 beads per milliliter of sample. To deplete 99% CD4 cells from the starting sample, the bead concentration must always be 2 x 10e7 beads per milliliter of sample. Please consult the package insert for recommended bead concentrations of each product.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Superparamagnetic means that the Dynabeads magnetic beads exhibit magnetic properties when placed within a magnetic field, but have no residual magnetism when removed from the magnetic field.
This means that your targeted cells, proteins, or nucleic acids are only subjected to magnetic forces during the time the beads are on the magnet. The beads do not aggregate, but remain evenly dispersed in suspension.
Find additional tips, troubleshooting help, and resources within our Dynabeads Cell Isolation and Expansion Support Center.
Yes. The antibodies are covalently bound and should be very stable in your applications.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Depending on the antibody coated on the Dynabeads magnetic beads, the shelf life can vary from 24-36 months.
Some kits may have 18 months shelf life depending on other components supplied in the kit. The kits are guaranteed for 6 months from when you receive them.
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center as well as our Protein Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), and Pulldown Support Center.
These two kits use different antibody clones which have different affinities for CD3 or CD28 antigen. The ratio of CD3/CD28 antibodies and bead concentrations are different as well in the two kits. Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Cat. No. 11131D) is mainly designed for T cell activation and Dynabeads Human T-Expander CD3/CD28 (Cat. No. 11141D) is mainly designed for T cell expansion.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Magnetic beads, unlike agarose beads, are solid and spherical, and antibody binding is limited to the surface of each bead. While magnetic beads do not have the advantage of a porous center to increase the binding capacity, they are significantly smaller than agarose beads (1 to 4 µm), which collectively gives them adequate surface area-to-volume ratios for optimum antibody binding.
High-power magnets are used to localize magnetic beads to the side of the incubation tube and out of the way to enable cell lysate aspiration without the risk of also aspirating immune complexes bound to the beads. Magnetic separation avoids centrifugation, which can break weak antibody-antigen binding and cause loss of target protein.
However, an issue with the use of magnetic beads is that bead size variations may prevent all beads from localizing to the magnet. Additionally, while immunoprecipitation using agarose beads only requires standard laboratory equipment, the use of magnetic beads for immunoprecipitation applications requires high-power magnetic equipment that can be cost-prohibitive. Read more about our Magnetic Immunoprecipitation Products (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/immunoprecipitation.html#products).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
The major differences are:
- Intended use: Dynabeads Human CD3/CD28/CD137 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. Dynabeads Human T Activator CD3/CD28 (without the CD137) is optimized for activation of naive T cells, which require a stronger anti-CD3 activation signal than Ag-specific/primed T cells.
- Different clones: The CD3 and CD28 antibodies of these two products are from different clones.
- Different protocols: There are very few beads required when activating Ag-specific/primed T cells (1 bead per 10 target cells) vs. naive T cells (1 bead per target cell), so follow the protocols for optimal results. Due to this, the bead concentration is also lower in the Dynabeads CD3/CD28/CD137 product. The solution will appear clear, and not as brown as the other Dynabeads magnetic beads, but, there are beads present! Mix the vial properly before taking out the required bead volume for any experiment.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Bead size for all of the T cell activation and expansion kits is 4.5 µm.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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)).
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Whether cells will internalize the Dynabeads magnetic beads during culture will depend on the cell type. Due to the bead size (usually 4.5µm in diameter) Dynabeads magnetic beads will not be internalized into the endocytic pathway e.g., via clathrin coated pits. The clathrin coated pits are typically not more than 500 nm in size, which is far too small for endocytosis of the beads. However, if cells with phagocytic activities (e.g., monocytes/macrophages) are present, the Dynabeads magnetic beads will be phagocytosed into the phagolysosomes by these specialized cells. So it would really depend on the cell type.
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We offer several Dynabeads magnetic beads that can be used for either positive isolation (keep the target cells) or for depletion (remove the target cell from a sample) that does not include any release mechanism:
- Dynabeads magnetic beads for depletion: Using Dynabeads magnetic beads for depletion is a very fast, efficient and easy method. Use pre-coated Dynabeads magnetic beads or coat your own target antibody onto our secondary coated beads, add to any sample (e.g., whole blood, PBMC, buffy coat, tissue digests), incubate for 20 minutes with mixing, apply to a magnet for 2 minutes, and you have your cells depleted.
- Dynabeads magnetic beads for positive isolation for molecular downstream assays: Positive isolation of target cells without bead release can be used when the aim is downstream molecular studies such as DNA, RNA, or protein analysis. In these applications, the isolated cells can be lysed while the beads are attached to the cells, and the beads can be removed after cell lysis. If the bead presence is not a problem, you can also culture the cells with the beads on. In most cases the surface antigen will be internalized after 2-3 days, and then the beads will fall off since the beads are too big to be internalized by the endocytosis pathway.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
In general, the size of the Dynabeads magnetic beads is so large that they will not be internalized. The clathrin-coated pits are typically not more than 500 nm in size, which will be too small for Dynabeads magnetic beads to be internalized by endocytosis. However, if the target cells have phagocytic activities such as monocytes/macrophages, the Dynabeads magnetic beads could be internalized by phagocytosis.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Thaw cells in their cryovial in a 37 degrees C water bath until a small ice-clump is left. Transfer the cells gently to a fresh 10-15 mL tube immediately after the cells are thawed and add 10 mL 20% FCS/human serum in droplets to the cells while gentle pipetting. Avoid air bubbles. Work fast. Centrifuge the cells 200 X g, 8 minutes. Discard the supernatant. Resuspend in the appropriate buffer/media.
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In general, freezing medium (10% DMSO and 90% FCS) or Gibco Recovery Cell Culture Freezing Medium (Cat. No. 12648-010) work well. Some cells will always die during the freezing process. In addition, freezing and thawing will cause some cells to lyse. The protocol to freeze mammalian cells using Gibco Recovery medium is as follows:
1. Thaw Recovery Cell Culture Freezing Medium, mix well, and keep at 2-8 degrees C until use.
2. For suspension cells proceed to step 3. For adherent cells, gently detach cells from the substrate on which they are growing using a suitable dissociation reagent such as Gibco TrypLE reagent. Resuspend cells in the complete medium required for that cell type.
3. Transfer cell suspension to a sterile 15 mL centrifuge tube.
4. Determine the viable cell density and percent viability using a Countess Automated Cell Counter (similar automated or manual methods may be used) and calculate the required volume of Recovery Cell Culture Freezing Medium to give a final cell density of 1 X 10E6 to 1 X 10E7 cells/mL.
5. Centrifuge cell suspension at 100-200 x g for 5-10 minutes. Aseptically decant supernatant without disturbing the cell pellet. Note: Centrifugation speed and duration may vary depending on cell type.
6. Resuspend the cell pellet in (2- 8 degrees C) chilled Recovery Cell Culture Freezing Medium at recommended viable cell density for specific cell type (typically 1 X 10E6 cells/mL or greater).
7. Dispense aliquots of cell suspension (mix frequently to maintain a homogeneous cell suspension) into cryovials according to the manufacturer's specifications (i.e., 1.5 mL in a 2 mL cryovial).
8. Achieve cryopreservation in an automated or manual controlled rate freezing apparatus following standard procedures (approximately 1 degree C decrease per minute).
9. Transfer frozen cells to liquid nitrogen, (vapor phase); storage at -200 degrees C to -125 degrees C is recommended.
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Bone marrow needs to be washed and diluted prior to addition of Dynabeads magnetic beads to make the sample less viscous. Washing and DNase treatment is recommended for preparing bone marrow cells prior to cell isolation using Dynabeads magnetic beads:
- Mix 2 mL (10E7-10E8 cells) bone marrow with 2 ml PBS w/ 0.1% BSA + 0.6% Na-citrate.
- Centrifuge at 600 g for 8 min at 18-25 degrees C.
- Discard the supernatant and resuspend to 5 mL with PBS w/ 0.1% BSA + 1mM CaCl2 + 0.5 mM MgCl2.
- Add 600 Kunitz units DNase I (120 Kunitz units DNase I per milliliter).
- Incubate cells for 30 minutes at 18-25 degrees C with both gentle tilting and rotation.
- Centrifuge cell suspension for 8 minutes, 600 x g, at 18-25 degrees C.
- Discard supernatant and resuspend cell pellet in 5 mL PBS w/ 0.1% BSA.
- Centrifuge cell suspension for 8 minutes, 600 x g, at 18-25 degrees C.
- Discard supernatant and resuspend at 1 x 10E8 cells per milliliter in RPMI 1640 / 1% FCS
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Follow standard tissue preparations using enzymes and mechanical disruption to get a single-cell suspension. Eliminate large aggregates by sieving the digested cell suspension through a cell strainer or filter through a 30 µm filter. Disruption of tissue normally results in some cell death and release of DNA. Free DNA will impair cell capture, recovery, and purity. DNase I treatment is performed by incubating the cell suspension in PBS with 0.1% BSA + 1 mM CaCl2 + 0.5 mM MgCl2 and 120 Kunitz units DNase I per ml at 18-25 degrees C for 30 min. (For CELLection products, wash cells to remove DNase before adding the beads.)
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Mononuclear cells (MNC), also known as peripheral blood mononuclear cells (PBMC), are prepared from whole blood, buffy coat, bone marrow, or umbilical cord blood by density gradient separation. The following protocol can be used for standard MNC preparation for positive isolation or depletion protocols:
1. Collect blood sample with anticoagulant present (EDTA, ACD, heparin). Dilute peripheral blood 1 + 1, buffy coat 1 + 2, bone marrow 1 + 1 and umbilical cord blood 1 + 3 in PBS w/ 0.1% BSA + 0.6% Na-citrate or 2 mM EDTA.
2. Layer up to 35 mL of the diluted sample over 15 mL gradient medium (such as Ficoll or Lymphoprep solution) in a 50 mL tube.
3. Centrifuge for 400 x g for 30-40 minutes at 18-20 degrees C. If blood has been stored for more than 2 hours, increase centrifugation time by 10 min.
4. Collect MNC from the interface and transfer cells to a 50 mL tube.
5. Wash MNC three times with PBS w/0.1% BSA by centrifugation at 300 x g for 8 min at 2-8 degrees C.
6. Resuspend the cells to 1 x 10E7 cells per milliliter in PBS with 0.1% BSA and cool to 2-8 degrees C.
Note: MNC contain T cells (50%), B cells (5-10%), NK cells (5-10%), and monocytes (30%) without granulocytes and very few platelets.
For use with Untouched/negative isolation kits, the following protocol is recommended to obtain MNC prep with low platelet numbers and the highest possible purity:
Whole blood/buffy coat and bone marrow can be used as a starting material.
1. Dilute 10-18 mL blood/buffy coat with PBS w/ 0.1% BSA + 0.6% Na-citrate or 2 mM EDTA to a total volume of 35 ml at 18-25 degrees C.
2. Add the diluted blood/buffy coat on top of 15 mL of gradient medium (such as Lymphoprep or Ficoll solution).
3 .Centrifuge at 160 x g for 20 min at 20 degrees C. Allow to decelerate without braking.
4. Remove 20 mL of supernatant to eliminate platelets.
5. Centrifuge at 350 x g for 20 min at 20 degrees C. Allow to decelerate without braking.
6 .Recover MNC from the plasma/Lymphoprep solution interface and transfer the cells to a 50 mL tube.
7. Wash MNC once with PBS w/ 0.1% BSA by centrifugation at 400 x g for 8 min at 2-8 degrees C.
8. Wash MNC twice with PBS w/ 0.1% BSA by centrifugation at 225 x g for 8 min at 2-8 degrees C and resuspend the MNC at 1 x 10E8 MNC per milliliter in PBS w/ 0.1% BSA.
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Buffy coat, also known as leukocyte concentrate, is the middle fraction of an anti-coagulated blood sample that sits under the plasma and on the top of red blood cells after centrifugation of the sample without using a density gradient reagent such as Ficoll solution. Buffy coat contains both leukocytes and platelets and can be used as a source of this cellular material.
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Typically, one milliliter of adult human blood contains:
~5 x 10E9 red blood cells
~7 x 10E6 leukocytes
~3 x 10E8 platelets
In the 7 x 10E6 leukocyte fraction, there are:
4 x 10E5 monocytes
1 x 10E5 NK cells
Lymphocytes:
2 x 10E5 B cells
1 x 10E6 T cells (approx. 70% are CD4+ T cells and 30% are CD8+ T cells)
Granulocytes:
5 x 10E6 neutrophils
2 x 10E5 eosinophils
4 x 10E4 basophils
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This will depend on your application. As a guideline, the 4.5 micron beads are best used for cell isolation and activation/expansion. These larger beads have a higher magnetic mobility, they are roughly the same size as mammalian cells, and are less likely to be taken up by the cells. The smaller 1 micron beads and 2.8 micron beads are often used when isolating nucleic acids or proteins, or for immunoprecipitation. In negative cell isolation kits, one micron beads are often used because of their higher binding capacity per milliliter of beads and faster binding kinetics. With negative selection, cells taking up any beads will not be a problem as you want to look at the remaining cell population anyway. The 2.8 micron Dynabeads magnetic beads, coated with secondary antibodies, protein A or protein G, or streptavidin are also used for positive cell isolation with primary antibodies of your own choice, targeting specific cell-surface antigens.
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Three different sizes of Dynabeads magnetic beads are available: One micron beads (look for MyOne magnetic beads in the product name), 2.8 micron beads, and 4.5 micron beads. In general, the binding capacity per milliliter of beads and binding kinetics increases as the bead size reduces.
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Dynabeads magnetic beads are super-paramagnetic, meaning they only display magnetic characteristics when a magnet is present. As soon as the magnet is removed, the beads handle like a liquid and are easily dispersed in the sample tube. For cell isolation purposes, this has clear advantages as it allows for gentle handing and reduced stress to the cells. Secondly, the beads all have the same size and shape, with rapid liquid-phase reaction kinetics. The smooth surface of the beads results in less non-specific binding. These properties tend to reduce variability and allow you to get more reliable and reproducible results for your purifications and your analyses whether you are looking at cells or any other target molecule (RNA/DNA/proteins/protein complexes/organelles/exosomes etc.)
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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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