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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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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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.