Magnetic Isolation of CD4+ CD25+ Regulatory T Cells from Human Mononuclear Cells (MNCs)

This protocol is intended for magnetic isolation of CD4⁺CD25⁺ regulatory T cells from human mononuclear cells (MNCs). The CD4⁺CD25- cell fraction can be used as effector cells in downstream inhibitory assays. The supplied protocol describes magnetic labeling and isolation from 1 x 10⁸ MNCs. In the first step the non-CD4⁺ MNC are labeled with Antibody Mix Human CD4. In the second step Depletion MyOne Dynabeads® are added to remove the non-CD4⁺ MNCs. In the third step Dynabeads® CD25 are added to the CD4⁺ T cells to capture the CD4⁺CD25⁺ T cells and in the last step Dynabeads® CD25 are removed from the cells.

Downstream Applications

Isolated cells may be used directly in any downstream application including flow cytometry and cell expansion 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) (f.ex. Gibco cat.no. 14190-094) supplemented with 0.1% BSA and 2mM EDTA (see Technical Recommendations for further information).
• Foetal Bovine Serum (FBS)
• Media: RPMI or equivalent w.1% FBS
• Mixer allowing both tilting and rotation.
• Magnet.
• Flow cytometry antibody reagents (optional). Recommended products and protocols.

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.
• This product should not be used with the Dynal® MPC-1 magnet (Cat. No. 120.01D)
• Never use less than recommended volume of Dynabeads®.
• Carefully follow the recommended pipetting volumes and incubation times.
• Avoid air bubbles during pipetting.
• Use primary fluorescent conjugated antibodies for flow cytometry. Avoid using secondary antibodies specific for mouse antibodies for flow cytometry staining.

Approximately 3–10% of the CD4⁺ T cell population expresses the CD25 antigen. This kit isolates highly pure CD4⁺ CD25⁺ regulatory T cells that express the intracellular transcription factor FOXP3. This protocol describes magnetic labelling and isolation of CD4⁺CD25⁺ regulatory T cells from 1x10⁸ human mononuclear cells (MNCs) using Dynabeads® Regulatory CD4⁺CD25⁺ T Cell Kit.

Preparations

• Isolate MNCs from anti-coagulated peripheral blood or leukocyte enriched buffy coat using standard procedure (see Technical Recommendations for further information).
• Prepare isolation buffer and RPMI w. 1% FBS.

Isolation procedure

When working with fewer cells than 1 x 10⁸, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly.

1. Resuspend 1 x 10⁸ MNCs in 500 μl isolation buffer and add 200 μl FBS and 200 μl Antibody Mix Human CD4. Mix well and incubate for 20 min at 2–8°C.


2. Add 10 ml cold isolation buffer to wash cells, followed by centrifugation for 8 min at 350xg.


3. Remove and discard the supernatant.


4. Add 2 ml cold isolation buffer to the cell pellet and resuspend.


5. Add 1 ml resuspended Depletion MyOne Dynabeads ®and mix well. Incubate for 15 min at room temperature under rolling and tilting.


6. Resuspend the bead-bound cells by vigorously pipetting 5 times using a pipette with a narrow tip opening (e.g., a 1000 μl pipette tip or a 5 ml serological pipette).


7. Add 3 ml of isolation buffer.


8. Place the tube in the magnet for 3 min.


9. Transfer the supernatant containing the bead-free CD4⁺ T cells to a new tube.


10. Repeat steps 8–9 with the tube containing the supernatant to remove residual Depletion MyOne Dynabeads®.


11. Spin down the cells for 8 min at 350xg and resuspend the cells in isolation buffer to a concentration of 1.5 x 10⁷⁺ cells/ml.


12. Add 200 μl Dynabeads® CD25 per 1,5 x 10⁷ CD4⁺ cells.


13. Mix well and incubate for 25 min at 2–8°C with rolling and tilting.


14. Place the tube in the magnet for a minimum of 1 minute. Carefully remove the supernatant containing the CD4⁺CD25- (effector) cells.


15. Remove the tube from the magnet and carefully resuspend the bead-bound cells in 5 ml isolation buffer by gently shaking the tube instead of pipetting the cells.


16. Place the tube in the magnet for a minimum of 1 min. Remove and discard the supernatant.


17. Wash one more time by carefully resuspending the cells in 5 ml isolation buffer and gently shaking the tube.


18. Place the tube in the magnet for a minimum of 1 minute. Remove and discard the supernatant.


19. Resuspend the bead-bound cells in 500 μl RPMI w.1% FBS


20. Add 80 μl DETACHaBEAD and incubate for 45 minutes at room temperature with tilting and rotation.


21. Place the tube in the magnet for a minimum of 1 minute. Carefully remove the supernatant containing the CD4⁺CD25⁺ cells to a new tube.


22. Wash the Dynabeads® CD25 twice in 1 ml RPMI w.1% FBS to obtain the residual cells and collect the supernatant after separation on a magnet.


23. Add 10 ml RPMI w.1% FBS to wash the cells, followed by centrifugation for 8 min at 350xg. Perform this washing step twice.


24. Discard the supernatant and resuspend the cells in a preferred cell culture medium. Learn more

Isolation buffer

PBS (phosphate buffered saline) from Gibco (Cat. No. 14190-094) supplemented with 0.1% BSA and 2 mM EDTA.
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 the Dynabeads® Regulatory CD4⁺CD25⁺ T Cell Kit.

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 x 10⁸ MNC per ml in isolation buffer.

For starting samples other than buffy coat please see recommended MNC preparation procedures. Please contact Invitrogen Dynal® for further technical information (see contact details).

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

Description of Materials
Depletion MyOne Dynabeads® are uniform, superparamagnetic polystyrene beads (1 μm diameter). Dynabeads® CD25 are uniform, superparamagnetic polystyrene beads (4.5 μm diameter). The Antibody Mix Human CD4 contains mouse IgG antibodies CD14, CD16 (specific for CD16a and CD16b), CD56, CDw123, CD36, CD8, CD19 and Glycophorin A. Supplied in PBS and 0.02% sodium azide (NaN₃).

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 .