Product Literature

Brochure: Immuno-oncology product resource guide

Manual / Product Insert

User Guide: CTS DynaMag Magnet

Version: B.0
Catalog #

Product FAQ

How many Dynabeads magnetic beads should I use for T cell activation or expansion?

Answer

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).

Answer Id: E12149

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Are the antibodies on your Dynabeads magnetic beads for Cell Isolation/Activation/Expansion covalently bound to the beads?

Answer

Yes. The antibodies are covalently bound and thus should be very stable in your applications.

Answer Id: E4656

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

How many Dynabeads magnetic beads should I use for T-cell expansion?

Answer

(1) For short-term stimulation of human T-cells (1-3 days): 1 bead per T cell

(2) For polyclonal expansion of T cells (more than 3 days): 3 beads per T cell

(3) For expansion of T-cell clones: 1 bead per 5-10 target cells

The use of Dynabeads CD3/CD28 beads will mainly induce a Th1 response involving IL-2, IFNg, and IL-12.

We have measured 10,000-20,000 pg per mL of IFNg and 60,000-80,000 pg per mL of IL-2 at 72 h after expansion using a Bio-Plex system.

Answer Id: E6214

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What are the major advantages of using CD3/CD28 Dynabeads magnetic beads to expand T cells?

Answer

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)).

Answer Id: E12142

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

How long should I stimulate my T cells with Dynabeads magnetic beads?

Answer

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).

Answer Id: E12146

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

When expanding my T cells, my cells are dying after activation with Dynabeads magnetic beads? Why is this?

Answer

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

Answer Id: E12077

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What is the bead size for Dynabeads T cell Activation or Expansion?

Answer

Bead size for all of the T cell activation and expansion kits is 4.5 µm.

Answer Id: E12143

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What should I consider when doing expansion of Ag-specific/primed T-cells?

Answer

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.

Answer Id: E12155

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

How do I remove the beads after the T cells are activated?

Answer

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.

Answer Id: E12151

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Is there a visible sign that my cells are activated after incubating with CD3/CD28-coated Dynabeads magnetic beads?

Answer

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.

Answer Id: E12152

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What Dynabeads magnetic bead size should I select?

Answer

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.

Answer Id: E12080

Was this answer helpful?

Yes
No
Thank you for your response

Product Literature

Application note: Cell culture quality control during T cell expansion