Isolate or Deplete CD3+ T Cells - Whole Blood, Buffy Coat, MNC Suspensions

Isolate or deplete CD3⁺ T cells directly from whole blood, buffy coat or MNC suspensions with Dynabeads CD3. For rapid and consistent results in protein or gene expression analysis, lyse the CD3⁺ T cells while they are still attached to the beads and directly process for further molecular analysis. Dynabeads CD3 can also be used for short term T cell activation.

Principle of Isolation

Dynabeads are mixed with the cell sample in a tube. The Dynabeads will bind to the target cells during a short incubation, and then the bead-bound cells are separated by a magnet.

• Positive Isolation - discard the supernatant and use the bead-bound cells for downstream applications (e.g. cell culture, protein or gene expression analysis).
  
• Depletion - discard the bead-bound cells and use the remaining, untouched cells for any application.

Description of Materials

Dynabeads CD3 are uniform, superparamagnetic, polystyrene beads (4.5 μm diameter) coated with a primary monoclonal antibody specific for the CD3 membrane antigen, which is predominantly expressed on human T cells.

Materials Supplied

• 5 ml Dynabeads CD3. 4 x 10⁸ beads/ml in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN₃).
• This product will process up to 2 x 10⁹ cells.

Additional Materials Required

• Magnet (Dynal MPC™): See Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps for magnet recommendations.
• Mixer allowing both tilting and rotation.
• Buffer 1: PBS w/0.1% BSA, pH 7.4
• Buffer 2: PBS w/0.1% BSA and 0.6% Nacitrate or 2 mM EDTA (without Ca²⁺ and Mg²⁺)
• Buffer 3: RPMI 1640 w/5% FCS or AB serum
• Density gradient medium for MNC preparation (1.077 g/l)

Dynabeads Washing Procedure

  1.    Resuspend the Dynabeads in the vial.

  2.    Transfer the desired volume of Dynabeads to a tube.

  3.    Add the same volume of Buffer 1, or at least 1 ml, and mix.

  4.    Place the tube in a magnet for 1 min and discard the supernatant.

  5.    Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of Buffer 1 as the initial volumen of Dynabeads.

Sample Preparation

Whole Blood and Buffy Coat

Most depletions and positive isolations can use whole blood and buffy coat as a starting sample. Buffy coat is 8-10 times more concentrated than whole blood with regard to number of leucocytes. For this product you have to wash the blood/buffy coat to remove interfering soluble factors.

1. Dilute the whole blood or buffy coat in Buffer 2 (1+2).


2. Centrifuge at 600 x g for 10 min at 2-8°C.


3. Discard the plasma fraction/upper layer.

Resuspend blood to the original volume in Buffer 2 and buffy coat 1+1 in Buffer 2 before adding the beads
Please visit the page Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species and follow our QuickLinks for recommended sample preparation procedures.

Cell Isolation

Critical Steps for Cell Isolation

• Use a mixer that provides tilting and rotation of the tubes to ensure Dynabeads do not settle at the bottom of the tube.
• When incubating Dynabeads and cells, the incubation temperature must be 2-8°C to reduce phagocytic activity and other metabolic processes.
• Never use less than 25 μl Dynabeads per ml cell samples.

Table 1: Volume of Dynabeads added per ml of cells. The volumes can be scaled up as required

Depletion or Positive Isolation of CD3+ T Cells

1. Add the appropriate volume of Dynabeads to the prepared sample. See table 1.


2. Incubate for 20 min (positive isolation) or 30 min (depletion) at 2 - 8°C with gentle tilting and rotation.


3. Place the tube in a magnet for 1 min.


4. For depletion, transfer supernatant to a new tube for further use.


5. For positive isolation, discard the supernatant and wash the bead-bound cells 3 times by resuspending in the original volume of Buffer 1, and separate using a magnet. For protein and gene expression studies, lyse cells while still attached to the beads and transfer supernatant to a new tube for further mRNA, protein or other subcellular isolations.

Detachment of Beads from Purified Cells

Dynabeads can be detached from the cells by incubating bead-bound cells in Buffer 3 for at least 6 hours at 37°C as described below.

1. Pipette the cell suspension to release beads.


2. Remove the detached Dynabeads by placing the tube on a magnet for 1 min and transfer the supernatant to a tube. The CD3 antigen will be down-regulated during incubation with beads but will be reexpressed on the cell surface after a short incubation period.

Direct Isolation and Stimulation of CD3⁺ T Cells

Isolate CD3⁺ T cells from a cell suspension with Dynabeads, and incubate bead-bound cells in a CO₂ incubator  at 37°C.

  1.    Isolate CD3⁺ T cells as described above

  2.    Resuspend the bead-bound cells at 1 x 10⁷ beads per ml in Buffer 3 and transfer directly to culture wells or flask.

Short-Term T Cell Activation

Dynabeads CD3 can be used for short-term activation of both CD4⁺ and CD8⁺ T cells. T cells can be stimulated directly in a mixed cell suspension, or individual T cell subsets can be stimulated after isolation using the
Dynal CD4 Positive Isolation Kit (Cat. no. 113.31), the CD8 Positive Isolation Kit (Cat. no. 113.33), CD4 T Cell Negative Isolation Kit (Cat. no. 113.17) or the CD8 T Cell Negative Isolation Kit (Cat. no. 113.19).

1. Isolate and detach CD4⁺ and CD8⁺ T cells according to the recommended protocols in the package inserts.


2. Resuspend the isolated cells in Buffer 3 to 1 x 10⁶ cells/ml.


3. Add 2.5 μl of washed Dynabeads CD3 per ml of cell suspension, (bead:cell=1:1)


4. Incubate the culture in a CO₂-incubator at 37°C for up to 3 days (longer incubation time will lead to apoptosis). For expansion of T cells, use Dynabeads CD3/CD28 T Cell Expander (Cat. no. 111.31/32)

1. Smith DM (2004) Liver transplantassociated Graft-versus-host Disease. ASHI Quarterly Sci. Comms.: First Quarter 12-13.


2. Majumdar MK et al. (2002) Characterisation and functionality of cell surface molecules on human mesenchyma  stem cells. J Biomed Sci. 228-241.


3. Dardalhon V et al. (2000) Highly efficient gene transfer in naive human T cells with a murine leukemia virus based vector. Blood. 96: 885-893.