Dynabeads™ Human T-Expander CD3/CD28

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.

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.