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MagCellect Mouse CD4+ CD25+ Regulatory T Cell Isolation Kit

Key Product Details

Assay Procedure

Refer to the product datasheet for complete product details.

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

Step 1

  • Incubate the single-cell suspension of splenocytes with the MagCellect Mouse 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

Step 2

  • Incubate the CD4+ T cell suspension with the MagCellect Anti-Mouse 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
 

 

Kit Components

Reagents Supplied in the MagCellect Mouse CD4+CD25+ Regulatory T Cell Isolation Kit (Catalog # MAGM208)

  • MagCellect Mouse CD4+ T Cell Biotinylated Antibody Cocktail; 1 mL in PBS containing BSA and preservative
  • MagCellect Streptavidin Ferrofluid; 1.5 mL in a solution containing BSA and preservative
  • Anti-Mouse CD25 Biotinylated Antibody; 0.5 mL in a solution containing BSA and preservative
  • MagCellect Plus Buffer (10X); 25 mL of a 10X concentrated buffer

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

 

 

Other Supplies Required
  • 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.

 

 

Procedure Overview

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

PBMC Preparation

Enrich for mononuclear cells by using a density gradient or any other method that provides a single cell suspension.

Wash the cells 2 times with excess PBS.

Centrifuge the cells for 10 minutes at 200 x g.

Enrich for mononuclear cells by using a density gradient or any other method that provides a single cell suspension.

Decant the supernatant. If necessary, remove red blood cells using R&D Systems Mouse Erythrocyte Lysing Kit (Catalog # WL2000).

Resuspend the cells in a small volume of cold 1X MagCellect Plus Buffer.

Decant the supernatant.

Perform a cell count.

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

Perform a cell count.
 

 

Step 1 - Negative selection of CD4+ T cells

Transfer 2 x 108 cells (1.0 mL) into a 5-mL polystyrene tube.

Add 200 μL of MagCellect Mouse CD4+ T Cell Biotinylated Antibody Cocktail.

Mix the cell-antibody suspension and incubate at 2 °C to 8 °C for 15 minutes.

Transfer 20 x 10 8 cells (1.0 mL) into a 5-mL polystyrene tube.

Add 250 μL of MagCellect Streptavidin Ferrofluid to the cell suspension.

Mix gently.

Incubate at 2 °C to 8 °C for 15 minutes.

Add 250 uL of MagCellect Streptavidin Ferrofluid to the cell suspension.

Add 1.6 mL of cold 1X MagCellect Plus Buffer.

Mix gently.

Add 1.6 mL of cold 1X MagCellect Plus Buffer.

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.

Place the reaction tube in the MagCellect Magnet.

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.

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

Add 10 µL of MagCellect Mouse CD25+ T Cell Biotinylated Antibody Cocktail per 1 x 107 cells.

Mix the cell-antibody suspension and incubate at 2 °C to 8 °C for 15 minutes.

Add 10 uL of MagCellect Mouse CD25+ T Cell Biotinylated Antibody Cocktail per 1 x 10 7 cells.

Add 250 µL of MagCellect Streptavidin Ferrofluid per 1 x 107 cells in the suspension.

Mix gently.

Incubate at 2 °C to 8 °C for 15 minutes.

Add 250 uL of MagCellect Streptavidin Ferrofluid per 1 x 10 7 cells in the suspension.

Add cold 1X MagCellect Plus Buffer to reach a volume of 1.0 mL.

Mix gently.

Add cold 1X MagCellect Plus Buffer to reach a volume of 1.0 mL.

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 1.0 mL cold 1X MagCellect buffer.

Place the reaction tube in the MagCellect Magnet.

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. Mouse Treg cell populations isolated by this negative and positive selection protocol typically have a purity of 84-94%.

To complete the cell isolation procedure, repeat Step 2 with the resuspended cell fraction.
 

 

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 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 magnetic separation step according to the following formula: 3 mL for each 20 x 107 cells processed. Also increase the magnetic incubation time described in to 8 minutes. Reaction volume adjustments must be made using 1X MagCellect Buffer just prior to the magnetic separation step.

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