Introduction

This product is intended for isolation of untouched human T cells from peripheral blood mononuclear cells (PBMC) by depleting B cells, NK cells, monocytes, platelets, dendritic cells, granulocytes and erythrocytes. Isolated T cells are bead- and antibody-free and are suitable for any downstream application.

Principle of Isolation

A mixture of mouse IgG antibodies against the non-T cells is added to the starting sample. Depletion Dynabeads® are added and will bind to the antibody-labeled cells during a short incubation. The bead-bound cells are subsequently separated on a magnet and discarded. The remaining, untouched human T cells can be used for any application.

Downstream Applications

Isolated T cells can be analyzed in flow cytometry and used in any downstream application e.g. T cell culture, T cell activation and expansion, functional assays, molecular studies.

Sample Preparation

Untouched T cells are isolated from a PBMC suspension. We recommend using a sample preparation procedur  that gives low platelet numbers. See Technical Recommendations, or visit Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species for recommended sample preparation procedures.

Additional Materials Required

  • Mixer allowing both tilting and rotation.
  • DynaMag™ magnet: See Magnets for Molecular and Cell Separation Applications for magnet recommendations.
  • Isolation buffer: Ca2+ and Mg2+ free phosphate buffered saline (PBS) (e.g. Gibco cat.no. 14190-094) supplemented with 0.1% BSA and 2mM EDTA.
  • Heat inactivated Fetal Bovine Serum (FBS)/Fetal Calf Serum (FCS).
  • Lymphoprep® for PBMC preparation (Axis Shield PoC, Norway).


Note:

  • Keep the buffers cold.
  • BSA can be replaced by human serum albumin (HSA) or 2% FBS/ FCS.
  • EDTA can be replaced by 0.6% sodium citrate.
  • PBS containing Ca2+ or Mg2+ is not recommended.


Critical notes

  • Depletion Dynabeads® should be washed before use (see ‘Dynabeads® Washing Procedure’)
  • Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads® do not settle in the tube
  • This product should not be used with Dynal® MPC™-1 (cat.no. 120.01D).
  • Follow the recommended volumes and incubation times.
  • Avoid air bubbles during pipetting

Protocol

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 Isolation buffer, 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 Isolation buffer as the initial volume of Dynabeads® (step 2).

Preparations

Prepare a PBMC suspension (low platelet numbers, see Technical Recommendations). Resuspend the suspension at 1 x 108 PBMC per ml in Isolation buffer.

Isolation Procedure

This protocol is based on 5 x 107 PBMC and is scalable from 1 x 107 - 5 x 108 cells according to table 1.
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Volumes per 1 x 107 PBMC Volumes per 5 x 107PBMCVolumes per 5 x 108 PBMC
Tube size3-5 ml15 ml50 ml
Cell volume (step 1)
100 μl
500 μl
5 ml
FBS/FCS (step 2)
20 μl
100 μl
1 ml
Antibody Mix (step 3)
20 μl
100 μl
1 ml
Washing (step 5)
2 ml
5 ml
40 ml
Resuspension (step 6)
100 μl
500 μl
5 ml
Depletion Dynabeads® (step 7)
100 μl
500 μl
5 ml
Volume added before
magnetic separation (step 10)
1 ml
5 ml
20 ml

Note: The difference in volumes for the small/medium/large scale protocol is based on getting good working volumes during incubation with beads.

  1. Transfer 500 μl (5 x 107) PBMC in Isolation buffer to a tube.

  2. Add 100 μl heat inactivated FCS.

  3. Add 100 μl of Antibody Mix.

  4. Mix well and incubate for 20 min at 2-8°C.

  5. Wash the cells by adding 5 ml Isolation buffer. Mix well by tilting the tube several times and centrifuge at 300 x g for 8 min at 2-8°C. Discard the supernatant.

  6. Resuspend the cells in 500 μl Isolation buffer.

  7. Add 500 μl pre-washed Depletion Dynabeads®.

  8. Incubate for 15 min at 18-25°C with gentle tilting and rotation.

  9. Resuspend the bead-bound cells by gently pipetting 5 times using a pipette with a narrow tip opening, (e.g.   1,000 μl pipette tip or a 5 ml serological pipette).

  10. Add 5 ml Isolation buffer. (When working with < 1 x 107 PBMC during incubation with beads, add Isolation buffer up to a total volume of 1 ml).

  11. Place the tube in the magnet for 2 min.

  12. Transfer the supernatant to a new tube.

  13. Repeat step 10-12.

  14. To remove any residual Dynabeads®, place the tube in the magnet for 2 min and transfer the supernatant to an new tube. The supernatant contains the negatively isolated human T cells.

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Technical Recommendations

Sample Preparation

Preparation of PBMC from buffy coat to obtain low platelet numbers:

  1. Dilute 10 - 15 ml buffy coat with PBS Isolation buffer to a total volume of 35 ml at 18-25°C (RT).

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

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

  4. Remove 18-20 ml of supernatant to eliminate platelets.

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

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

  7. Wash PBMC once with Isolation buffer by centrifugation at 400 x g for 8 min at 2-8°C.

  8. Wash PBMC twice with Isolation buffer by centrifugation at 225 x g for 8 min at 2-8°C and resuspend the PBMC at 1 x 108 PBMC per ml in Isolation buffer.
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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. Chiang KP et al (2004) Reduced cellular expression and activity of the P129T mutant of fatty acid amide hydrolase: evidence for a link between defects in the endocannabinoid system and problem drug use. Human Mol. Gen. 13: 2113-2119.

  2. Falk M et al (2004) Caspase inhibition blocks human T cell proliferation by suppressing appropriate regulation of IL-2, CD25 and cell cycle-associated proteins.

  3. Harriague J and Bismuth G (2002) Imaging antigen-induced P13K activation in T cells. Nature Immunol. 3: 1090-1096.

  4. Mandrekar P et al (2004) Inhibition of myeloid dendritic cell accessory cell function and induction of T cell anergy by alcohol correlates with decreased IL-12 production. J. Immunol. 173: 3398-3407.

  5. Sato K et al (2005) TRAIL-transduced dendritic cells protect mice from acute graftversus-host disease and leukaemia relapse. J. Immunol. 174: 4025-4033.

  6. Wang F et al (2006) Prominent Production of IL-20 by CD68+/CD11c+ Myeloid-Derived Cells in Psoriasis: Gene Regulation and Cellular Effects. J Inv. Derm. 126: 1590-1599.

  7. Ghadiri A et al (2007) Critical function of Ikaros in controlling Aiolos gene expression. FEBS Letters. 581: 1605-1616.

  8. Haider AS et al (2007) Cellular genomic maps help dissect pathology in human skin disease. J Inv. Derm. 128: 606-615.
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113.44D.indd     Rev 001     5-May-2008