Introduction

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)

Ordering Information

Catalog # Name Size List Price (USD) Qty
11151D Dynabeads® CD3 5 mL 910.00

Protocols

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

Positive isolation Depletion
Sample volume -
  • 1 x 107 MNC*/ml
  • washed whole blood
  • washed buffy coat
1 ml
1 ml
Volume of Dynabeads 25 μl 50 μl
Total no. of cells
processed per product
2 x 109 cells
1 x 109 cells


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

I
solate 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)


General Information

Invitrogen Dynal AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Storage/Stability

This product is stable until the expiry date stated on the label when stored unopened at 2-8°C. Store opened vials at 2-8°C and avoid bacterial contamination. Keep Dynabeads in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.

Warnings And Limitations

This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide as a preservative, which is cytotoxic.

Avoid pipetting by mouth!

Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at .
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References

  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.


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111.51D.indd   Rev 002    5-May-2007