Introduction

This protocol is intended for positive magnetic isolation of CD3+ T cells from human peripheral blood mononuclear cells (PBMCs). In the first step, FlowComp™ Human CD3 Antibody is added and will bind to the target cells. In the second step, CD3+ T cells, that have bound the specific antibodies, are captured by the Dynabeads®. In the third and last step, the cells are released from the Dynabeads®.

Downstream Applications

Isolated cells are bead-free and may be used directly in any downstream application including flow cytometry. The cells readily proliferate in response to Dynabeads® CD3/CD28 T Cell Expander (Invitrogen Dynal® Cat. no.111.31D/111.32D) and can be measured by incorporation of EdU (e.g. EdU Click-iT, Invitrogen Cat. no A10202) or in a CFSE assay (Invitrogen Cat. no. 34554).  See recommended products and protocols.

Additional Materials Required

Materials that are not included, but are needed to perform the entire protocol:

  • Isolation Buffer: Ca2+ and Mg2+ free phosphate buffered saline (PBS) (e.g. Invitrogen Gibco Cat. no.14190) supplemented with 0.1% BSA and 2mM EDTA. BSA can be replaced by human serum albumin (HSA) or 2% FBS/FCS.
  • Mixer allowing both tilting and rotation.
  • Dynal® Magnet: See magnet recommendations.
  • Optional: Flow cytometry antibody reagents. Invitrogen recommends using anti-CD3 clone UCHT-1 from CALTAG as primary fluorescent antibody for flow staining of cells after isolation (available conjugated to different fluorochromes, e.g Invitrogen Cat. no. CD0304). Optional clones: OKT3, HITa3. See recommended products and protocols
  • Optional: For viability analysis, SYTOX® Red (Invitrogen Cat. no. S34859) is recommended.


Critical notes

  • Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads® do not settle in the tube.
  • Resuspend the Dynabeads® in the vial carefully before use, i.e. vortex for >30 sec., or tilt and rotate for 5 minutes. This product should not be used with Dynal® MPC™-1 (Invitrogen Dynal® Cat. no. 120.01D)
  • Avoid air bubbles during pipetting.
  • Never use less than recommended volume of Dynabeads®.
  • Carefully follow the recommended pipetting volumes and incubation times.
  • Keep all buffers cold

Protocol

In PBMCs form normal blood donors, approximately 60–70% of the cells express CD3. This protocol describes magnetic capture and isolation of highly pure CD3+ T cells from 5 × 107 PBMCs using Dynabeads® FlowComp™ Human CD3. When working with fewer cells than 5 × 107, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly.

Preparations

  • Isolate PBMCs from anti-coagulated peripheral blood or leukocyte enriched buffy coat using standard procedure (see Technical Advise for further information).
  • Prepare a single cell suspension of 1 × 108 cells/ml in isolation buffer.
  • Prepare approximately 10 ml of Isolation Buffer per 5 × 107cells.

Isolation Procedure

All incubations at room temperature (RT) can also be performed at 2–8°C.

  1. Use 500 μl (5 x 107 cells) from the preparation above, resuspend and add 25 μl FlowCompTM Human CD3 antibody.

  2. Mix well and incubate for 10 minutes at 2–8°C.

  3. Add 2 ml Isolation Buffer to wash cells, followed by centrifugation for 8 minutes at 350 × g.

  4. Remove and discard the supernatant.

  5. Add 1 ml Isolation Buffer to the cell pellet and resuspend.

  6. Add 75 μl resuspended FlowComp™ Dynabeads® and mix well.

  7. Incubate for 15 min at RT under rolling and tilting.

  8. Place the tube on the magnet for minimum 1 minute. Carefully remove and discard the supernatant.

  9. Remove the tube from the magnet. Add at least 1 ml Isolation Buffer and resuspend the bead-bound cells by gentle pipetting 5 times.

  10. Place the tube on the magnet for minimum 1 minute. Carefully remove and discard the supernatant.

  11. Remove the tube from the magnet and carefully resuspend the bead-bound cells in 1 ml FlowComp™ Release Buffer.

  12. Incubate for 10 minutes at RT under rolling and tilting.

  13. Mix the cells by pipetting 10 times and place the tube in the magnet for 1 minute.

  14. Transfer the supernatant containing the bead-free cells to a new tube, and again place on the magnet for 1 minute to remove any residual beads.

  15. Transfer supernatant containing the bead-free cells to a new tube.

  16. Add 2 ml Isolation Buffer followed by centrifugation for 8 minutes at 350 x g.

  17. Discard the supernatant and resuspend the cell pellet in preferred medium.

Keep the cells on 2–8°C until further use in downstream applications.


TOP

Technical Recommendations

General Troubleshooting

To avoid unspecific labeling of cells during flow staining, we recommend to use gammaglobulin prior to staining with primary fluorescent antibody.

For better purity, repeat the washing step once or transfer the bead-bound cells to a new tube before adding the
FlowComp™ Release Buffer

Preparation of PBMC from Buffy Coat

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

  • 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.
  • Add the diluted buffy coat on top of 15 ml of Lymphoprep™.
  • Centrifuge at 160 × g for 20 minutes at 20°C. Allow to decelerate without brakes.
  • Remove 20 ml of supernatant to eliminate platelets.
  • Centrifuge at 350 × g for 20 minutes at 20°C. Allow to decelerate without brakes.
  • Recover PBMC from the plasma/Lymphoprep ™ interface and transfer the cells to a 50 ml tube.
  • Wash PBMC once with PBS with 0.1% BSA by centrifugation at 400 × g for 8 minutes at 2–8°C.
  • Again wash PBMC with PBS with 0.1% BSA by centrifugation at 225 × g for 8 minutes at 2–8°C and resuspend the cells at 1 × 108 PBMC per ml in PBS w/0.1% BSA.


For starting samples other than buffy coat, please see recommended PMBC preparation procedures.

For further technical information contact Dynal®.

General Information

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

Description of Materials

Dynabeads® FlowComp™ are uniform, superparamagnetic beads (2.8 μm in diameter). Supplied at a concentration of approx. 1 × 109 beads (10 mg) per ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin (BSA) and 0.02% sodium azide (NaN3) as pereservatives.

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 .
TOP
LT112