Introduction

This protocol is intended for positive magnetic isolation or depletion of CD25high T cells from human samples such as mononuclear cells (MNCs) or whole blood. Dynabeads® bind to the target cells during a short incubation, and then the bead-bound cells are separated by a magnet.

Depletion
– discard the bead-bound cells and use the remaining, untouched cells for any application.

Positive isolation – discard the supernatant and use the bead-bound cells for downstream molecular applications.

This protocol describes isolation from up to 2.5x107 MNCs. Isolated cells are ideal for use in downstream molecular assays such as protein and gene expression analysis.

Downstream Applications

Isolated cells may be lysed while attached to the beads and directly processed for further molecular analysis. For recommended products and protocols visit the Immunology Research Guide page.

Additional requirements


Critical notes

  • Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads® do not settle at the bottom of the tube.
  • Never use less than recommended volume of Dynabeads®.
  • Carefully follow the recommended pipetting volumes and incubation times.
  • Avoid air bubbles during pipetting.

Protocol

Approximately 1x106 T cells are present per ml human blood, and about 5–10% of these T cells strongly express the CD25 antigen. This protocol describes depletion or positive isolation of CD25high T cells from 2.5x107 human mononuclear cells (MNCs) using Dynabeads® CD25.

For scale-up to larger cell numbers, adjust the volumes accordingly.

Preparations

  • Start with whole blood or isolate MNCs from anti-coagulated peripheral blood or leukocyte enriched buffy coat using standard procedure (see Technical Support for further information).
  • Prepare approximately 6 ml of isolation buffer per 2.5x107cells.


Dynabeads® Washing Procedure

Dynabeads® should be washed before use.

  • Resuspend the Dynabeads® in the vial.
  • Transfer the desired volume of Dynabeads® to a tube.
  • Add the same volume of Isolation buffer, or at least 1 ml, and mix.
  • Place the tube in a magnet for 1 min and discard the supernatant.
  • Remove the tube from the magnet and resuspend the washed Dynabeads® in the same volume of Isolation buffer as the initial volume of Dynabeads®  (bullet point 2).


Depletion or Positive Isolation of CD25+ cells


  • Resuspend 2.5x107 MNCs in 1 ml Isolation buffer and add 25 μl (positive isolation) or 50 μl (depletion) washed Dynabeads® CD25.
  • Incubate for 20 min (positive isolation) or 30 min (depletion) at 2 –8°C with gentle tilting and rotation.
  • Place the tube in a magnet for 2 min.
  • For depletion, transfer the supernatant to a new tube for further use.
  • For positive isolation, discard the supernatant and wash the beadcaptured cells 3 times by resuspending in Isolation buffer to the original sample volume, and separate using a magnet.
  • Keep the cells on 2–8°C until further use in downstream applications. 


For rapid and consistent results in protein or gene expression analysis, lyse the CD25+ cells while they are still attached to the beads and directly process for further molecular analysis.For further technical advice please visit Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species. 

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 .

Technical Support

Isolation buffer

PBS (phosphate buffered saline) supplemented with 0.1% BSA and 2mM EDTA, e.g. Gibco cat.no. 14190-094.
If preferred, PBS with 2% fetal calf serum and 1 mM EDTA may be used.

Preparation of MNC from Buffy Coat

This method gives low platelet numbers and is recommended for use with Dynabeads® products.

  1. Dilute 10–18 ml buffy coat with PBS with 0.1% BSA + 0.6% Na-citrate or 2 mM EDTA to a total volume of 35 ml at 18–25°C.

  2. Add the diluted buffy coat on top of 15 ml of Lymphoprep™.

  3. Centrifuge at 160 x g for 20 min at 20°C. Allow to decelerate without brakes.

  4. Remove 20 ml of supernatant to eliminate platelets.

  5. Centrifuge at 350 x g for 20 min at 20°C. Allow to decelerate without brakes.

  6. Recover MNC from the plasma/Lymphoprep interface and transfer the cells to a 50 ml tube.

  7. Wash MNC once with PBS with 0.1% BSA by centrifugation at 400 x g for 8 min at 2–8°C.

  8. Wash MNC twice with PBS with 0.1% BSA by centrifugation at 225 x g for 8 min at 2–8°C and resuspend the MNC at 2,5 x 107 MNC per ml in Isolation buffer.


For starting samples other than buffy coat please see the page Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species for recommended MNC preparation procedures. Please contact Invitrogen Dynal®  for further technical information (see contact details).

References

  1.    Jiang S et al (2003) Induction of allopeptide-specific human CD4+CD25+ regulatory T cells ex vivo. Blood 102: 2180-2186.

  2.    Guyot-Revol V et al (2006) Regulatory T Cells are expanded in blood and disease sites in patients with tuberculosis. AJRCCM 173: 803-810.


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111.57D.indd    Rev 002     5-May-2007