CD4+ CD25+ Regulatory T Cell Isolation from PBMC or Splenocyte Preparations by Magnetic Selection

This protocol provides a basic guide for the magnetic selection of CD4⁺ CD25⁺ Regulatory T lymphocytes from preparations of peripheral blood mononuclear cells (PBMC) or splenocytes. Isolation of CD4⁺ CD25⁺ regulatory T cells is performed using a combination of negative and positive selection methods.

In Step 1, a mononuclear cell suspension is incubated with a biotinylated antibody cocktail which targets unwanted cells. Streptavidin ferrofluid is added to the reaction, and the streptavidin coated nanoparticles interact with the biotinylated antibody tagged cells. The tube containing the cell suspension is then placed within a magnetic field. Magnetically tagged cells (unwanted cell fraction) will migrate toward the magnet, leaving the untagged cells (desired cell population) in suspension to be harvested by aspiration while the tube remains in the magnetic field.

In Step 2, the negatively isolated CD4⁺ T cells from Step 1 are subjected to positive selection. The cells are tagged with an anti-CD25 biotinylated antibody followed by addition of streptavidin ferrofluid. The tube containing the cell suspension is placed in the magnet. Magnetically tagged cells will migrate toward the tube wall on the magnet side (desired cell population), leaving the untagged (unwanted) cells in suspension. Unwanted cells are removed by aspiration while the tube remains in the magnet. The tube containing the CD4⁺ CD25⁺ Regulatory T cells is then removed from the magnet. The enriched cells are available for a variety of applications including tissue culture, immune status monitoring, and flow cytometry.

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Why Isolate CD4⁺ T Cells?

Regulatory T cells (Treg), are a minor subset of CD4⁺ T cells that can suppress the development and progression of immune reactions. They also play an important role in the balance between tolerance and autoimmunity. Treg cells are characterized by the expression of CD4 and CD25 on the cell surface and the intracellular transcription factor FoxP3. The isolation of regulatory T cells facilitates research into their critical role in immune system regulation.

Preparation of PMBC and Splenocytes

PBMC and splenocyte suspensions are suitable starting samples for the isolation of CD4⁺ CD25⁺ regulatory T cells. Follow the instructions in this protocol to obtain PBMC and splenocytes.

Isolation of CD4⁺ CD25⁺ Regulatory T Cells from PBMC or Splenocytes

MagCellect™ CD4⁺ CD25⁺ regulatory T cell isolation kits are designed to isolate CD4⁺ CD25⁺ regulatory T cells via both negative and positive selection principles. MagCellect kits are available for the isolation of (human or mouse CD4⁺ CD25⁺ regulatory T cells. These kits contain sufficient quantities of the following reagents to process 1 x 10⁹ total cells:

CD4⁺ T cell biotinylated antibody cocktail
Anti-CD25 biotinylated antibody
MagCellect Streptavidin Ferrofluid
MagCellect Plus Buffer (10X)

Other Materials Required

Sterile Pasteur pipettes or transfer pipettes
Hemocytometer
Ficoll-Hypaque™
MagCellect Magnet (Catalog # MAG997) or equivalent
12 x 75 mm (5 mL) or 17 x 100 mm (15 mL) polystyrene round-bottom tubes

Other Equipment Required

Centrifuge
Microscope

Step 1. CD4⁺ T Cell Selection Procedure

This procedure is for processing 2 x 10⁸ total cells using 5 mL tubes and the MagCellect Magnet. For processing other quantities of cells please refer to the Technical Hints section of this protocol. Cells and reagents should be kept cold using an ice bath or a refrigerator. Reaction incubations must be carried out at 2-8 °C in a refrigerator and not in an ice bath to avoid excessively low temperatures that can slow the kinetics of the optimized reactions.

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Prepare 10 mL of 1X Buffer for each 2 x 10⁸ cells to be processed by mixing 1.0 mL of 10X MagCellect Plus Buffer with 9.0 mL sterile deionized or distilled water. The 1X Buffer should be kept on ice or refrigerated and used within 24 hours.

Prepare a single cell suspension of human leukocytes by following the Leukocyte Preparation Protocol. Cells must be suspended in cold 1X Buffer prior to beginning the procedure and be at a cell density of 2 x 10⁸ cells/mL (human) or 1 x 10⁸ cells/mL (mouse).

Transfer 2 x 10⁸ cells (2.0 mL volume) into a 5 mL polystyrene tube. Add 200 µL of CD4⁺ T Cell Biotinylated Antibody Cocktail. Gently mix the cell-antibody suspension, avoiding bubble formation, and incubate at 2-8 °C in a refrigerator for 15 minutes.

Add 250 µL of streptavidin ferrofluid to the cell suspension, mix gently, and incubate at 2-8 °C in a refrigerator for 15 minutes.

At the end of the incubation period, bring the volume of the reaction in the tube to 3 mL by adding 0.55 mL (human) or 1.6 mL (mouse) of 1X Buffer. Mix gently to ensure that all reactants in the tube are in suspension.

Place the reaction tube in the MagCellect Magnet that has been positioned horizontally to accommodate 5 mL tubes and incubate for 6 minutes at room temperature. Magnetically tagged cells will migrate toward the magnet (these are the unwanted cells), leaving the untouched desired cells in suspension in the supernatant.

Recovery of CD4⁺ T cells is achieved as follows: While the tube is in the magnet, use a sterile Pasteur pipette or transfer pipette to carefully aspirate all of the reaction supernatant and place it in a new 5 mL tube. Remove the tube containing the magnetically trapped cells from the magnet and discard.

To ensure that all of the magnetic nanoparticles have been removed, repeat the magnetic depletion (steps 6 and 7) with the new tube containing the recovered cells. The supernatant obtained at the end of these steps is the final depleted cell fraction containing the enriched CD4⁺ T cells. The cells are now ready for anti-CD25 based selection as described in Step 2.

Step 2. CD4⁺ CD25⁺ T Cell Selection Procedure

Transfer counted CD4⁺ T cells into a 15 mL polystyrene tube. Wash cells in cold 1X Buffer. Resuspend cells with 100 µL cold 1X Buffer per 1 x 10⁷ cells and transfer the cell suspension to a 5 mL tube.

Add 10 µL of anti-CD25 biotinylated antibody per 1 x 10⁷ cells. Gently mix the cell-antibody suspension, avoiding bubble formation, and incubate at 2-8 °C in a refrigerator for 15 minutes.

Add 25 µL (human) or 10 µL (mouse) of streptavidin ferrofluid per 1 x 10⁷ cells to the cell suspension, mix gently, and incubate at 2-8 °C in a refrigerator for 15 minutes.

At the end of the incubation period, bring the volume of the reaction in the tube to 1 mL by adding 1X Buffer. Mix gently to ensure that all reactants in the tube are in suspension.

Place the reaction tube in the MagCellect Magnet that has been positioned horizontally to accommodate 5 mL tubes and incubate for 6 minutes at room temperature. Magnetically tagged cells will migrate toward the magnet (these are the desired CD4⁺ CD25⁺ T cells), leaving the untagged undesired cells in suspension in the supernatant.

Removal of unwanted cells is achieved as follows: While the tube is in the magnet, use a sterile Pasteur pipette or transfer pipette to carefully aspirate all of the reaction supernatant and discard.

Remove the tube containing the magnetically trapped (wanted) cells from the magnet and resuspend the cells by adding 1 mL cold 1X Buffer.

To complete the isolation procedure, repeat the magnetic depletion (steps 5 and 6) one more time with the recovered cells.

Remove the tube containing the magnetically trapped (wanted) cells from the magnet, and resuspend cells by adding 0.5-1.0 mL of 1X Buffer or tissue culture media. This final magnetically isolated fraction contains the desired enriched CD4⁺ CD25⁺ cells. The cells are now ready to be used in downstream applications.

Data Examples

Enrichment of CD4+ T Cells and CD4+CD25+ Cells

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Enrichment of CD4⁺ T Cells (Step 1) and CD4⁺ CD25⁺ Regulatory T Cells (Step 2) from Peripheral Blood Mononuclear Cells Using the MagCellect Human CD4⁺ CD25⁺ Regulatory T Cell Isolation Kit. Following isolation of CD4⁺ T cells (left) and CD4⁺CD25⁺ regulatory T cells (right) using the MagCellect Human CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Catalog # MAGH104), cells were stained with FITC-conjugated Mouse Anti-Human CD4 Monoclonal Antibody (Catalog # FAB3791F) and PE-conjugated Mouse Anti-Human CD3 epsilon Monoclonal Antibody (Catalog # FAB100P) provided in this kit.

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Enrichment of CD4⁺ CD25⁺ Regulatory T cells from Mouse Splenocytes Using the MagCellect Mouse CD4⁺ CD25⁺ Regulatory T Cell Isolation Kit. Following isolation of CD4+CD25+ regulatory T cells using the MagCellect Mouse CD4+CD25+ Regulatory T Cell Isolation Kit (Catalog # MAGM208), cells were stained using a FITC-conjugated Rat Anti-Mouse CD4 Monoclonal Antibody (Catalog # FAB554F) and a PE-conjugated Rat Anti-Mouse CD25 Monoclonal Antibody (Catalog # FAB2438P).

Technical Hints

If sterile cells are required following the cell selection, the entire procedure should be carried out in a laminar flow hood to maintain sterile conditions. Use sterile equipment when pipetting reagents that will be reused at a later date.
Avoid antibody capping on cell surfaces and non-specific cell tagging by working fast, keeping cells and solutions cold through the use of pre-cooled solutions, and by adhering to the incubation times and temperatures specified in the protocol. Increased temperature and prolonged incubation times may lead to non-specific cell labeling, thus lowering cell purity and yield.
When processing different numbers of cells, observe the following guidelines: keep antibody cocktail and ferrofluid incubation times and temperatures the same; keep the cell density at 1 x 10⁸ cells/mL; add 10 µL of the antibody cocktail per 1 x 10⁷ cells being processed; add 12.5 µL of streptavidin ferrofluid per 1 x 10⁷ cells being processed.
When processing 2 x 10⁸ cells or fewer, use the 12 x 75 mm (5 mL) tubes with the MagCellect Magnet horizontally positioned to accommodate up to six 5 mL tubes. Do not process more than 2 x 10⁸ cells in each 5 mL tube and do not exceed a total reaction volume of 3 mL in each tube. A reaction volume of 2 mL is recommended for processing 1 x 10⁸ cells. A reaction volume of 1 mL is recommended when processing 5 x 10⁷ or fewer cells. Reaction volume adjustments must be made using 1X Buffer just prior to the magnetic separation step.
When processing greater than 2 x 10⁸ cells, use 17 x 100 mm (15 mL) tubes with the MagCellect Magnet vertically positioned to accommodate up to two 15 mL tubes. Do not process more than 6 x 10⁸ cells in each 15 mL tube and do not exceed a total reaction volume of 9 mL in each tube. When using this larger tube, increase the reaction volume before the magnetic separation step according to the following formula: 3 mL for each 2 x 10⁸ cells processed. Also increase the magnetic incubation time described in step #6 to 8 minutes. Reaction volume adjustments must be made using 1X Buffer just prior to the magnetic separation step.