MagCellect Human CD4+ CD25+ Regulatory T Cell Isolation Kit

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, human CD4⁺ regulatory T cells can be isolated from PBMC using the following procedure:

Step 1

• Incubate the single-cell suspension of PBMC with the MagCellect Human CD4⁺ T Cell Biotinylated Antibody Cocktail
• Add the MagCellect Streptavidin Ferrofluid
• Place the tube in the MagCellect Magnet
• Collect the CD4⁺ cells while undesired cells remain attracted to the magnet

View Step 1
Graphic Procedure

 

Step 2

• Incubate the CD4⁺ T cell suspension with the MagCellect Anti-Human CD25 Biotinylated Antibody
• Add the MagCellect Streptavidin Ferrofluid
• Place the tube in the MagCellect Magnet
• While the tube is in the magnet, remove the unwanted cells from the tube
• Remove the tube from the magnet and resuspend the CD4⁺CD25⁺ T cells in buffer or media for counting

View Step 2
Graphic Procedure

 

Reagents Supplied in the MagCellect Human CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Catalog # MAGH104):

• MagCellect Human CD4⁺ T Cell Biotinylated Antibody Cocktail; 1 mL in PBS containing BSA
• MagCellect Streptavidin Ferrofluid; 3.0 mL in a solution containing BSA and preservative
• Anti-Human CD25 Biotinylated Antibody; 0.5 mL in a solution containing BSA and preservative
• MagCellect Plus Buffer (10X); 25 mL of a 10X concentrated buffer
• MagCellect Staining Reagent for Human CD4⁺CD25⁺ Regulatory T cells; 1 mL mixture of Anti-Human CD4-FITC and Anti-Human CD25-PE sufficient to perform 50 tests at 20 µL per test

This kit contains sufficient reagents to process up to 1 x 10⁹ total cells.

• MagCellect Magnet (Catalog # MAG997) or equivalent
• 12 x 75 mm (5 mL) or 17 x 100 mm (15 mL) polystyrene round bottom tubes
• 15 mL conical centrifuge tubes and benchtop centrifuge
• Sterile Pasteur pipettes or transfer pipettes

NOTE:Reaction incubations must be carried out at 2 to 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.

R&D Systems Protocol for the Magnetic Isolation of Human Regulatory T Cells

PBMC Preparation

 

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• Decant the supernatant. If necessary, remove red blood cells using R&D Systems Human Erythrocyte Lysing Kit (Catalog # WL1000).
• Resuspend the cells in a small volume of cold 1X MagCellect Plus Buffer.

 

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• Perform a cell count.
• Adjust the cell concentration to 1 x 10⁸ cells/mL with cold 1X MagCellect Plus Buffer.

 

Step 1 - Negative selection of CD4⁺ T cells

 

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• Add 250 μL of MagCellect Streptavidin Ferrofluid to the cell suspension.
• Mix gently.
• Incubate at 2 °C to 8 °C for 15 minutes.

 

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• Add 0.55 mL of cold 1X MagCellect Plus Buffer.
• Mix gently.

 

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• Place the reaction tube in the MagCellect Magnet.
• Incubate for 6 minutes at room temperature (18 °C to 25 °C).
• Transfer the supernatant containing the CD4⁺ T cells into a new 5 mL tube.

 

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• Repeat the magnetic depletion with the new tube containing the recovered cells. The supernatant obtained at the end of this step is the final depleted cell fraction containing the desired enriched CD4⁺ T cells.

 

Step 2 - Positive selection for CD4⁺CD25⁺ T cells

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• Add 250 μL of MagCellect Streptavidin Ferrofluid per1 x 10⁷ cells in the suspension.
• Mix gently.
• Incubate at 2 °C to 8 °C for 15 minutes.

 

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• Add cold 1X MagCellect Plus Buffer to reach a volume of 1 mL.
• Mix gently.

 

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• Place the reaction tube in the MagCellect Magnet.
• Incubate for 6 minutes at room temperature (18 °C to 25 °C).
• Remove unwanted cell suspension from the tube.
• Remove tube from the magnet and resuspend the CD4⁺CD25⁺ T cells in 1 mL cold 1X MagCellect buffer.

 

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• To complete the cell isolation procedure, repeat Step 2 with the resuspended cell fraction.
• The cells are now ready for counting and further downstream applications. Human Treg cell populations isolated by this negative and positive selection protocol typically have a purity of 85 – 95%.

 

1. Enrich for mononuclear cells by using a density gradient or any other method that provides a single cell suspension.
2. Wash the cells 2 times with excess PBS.
3. Centrifuge the cells for 10 minutes at 200 x g.
4. See Details
5. Transfer 2 x 10⁸ cells (2.0 mL) into a 5 mL polystyrene tube.
6. Add 200 μL of MagCellect Human CD4⁺ T Cell Biotinylated Antibody Cocktail.
7. Mix the cell-antibody suspension and incubate at 2 °C to 8 °C for 15 minutes.
8. See Details
9. Add 10 μL of MagCellect Human CD25⁺ T Cell Biotinylated Antibody Cocktail per 1 x 10⁷ cells.
10. Mix the cell-antibody suspension and incubate at 2 °C to 8 °C for 15 minutes.
11. See Details

• If sterile cells are required following 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 closely following 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 population 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 10 x 10⁷ 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 20 x 10⁷ 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 20 x 10⁷ 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 10 x 10⁷ 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 MagCellect Buffer just prior to the magnetic separation step.
• When processing greater than 20 x 10⁷ cells, use the 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 60 x 10⁷ 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 separation step using the following formula: 3 mL for each 20 x 10⁷ cells processed. Also increase the incubation time in the MagCellect Magnet to 8 minutes. Reaction volume adjustments must be made using 1X MagCellect Buffer just prior to the magnetic separation step.

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