MagCellect Mouse Naive CD4+ T Cell Isolation Kit

Refer to the product datasheet for complete product details.

View Graphic Procedure

The MagCellect Mouse CD4⁺ T Cell Isolation Kit (Catalog # MAGM205) contains the following reagents:

• MagCellect Mouse CD4⁺ T Cell Biotinylated Antibody Cocktail
• MagCellect Streptavidin Ferrofluid
• MagCellect Plus Buffer (10X)

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

Reagents

• Ficoll-Hypaque™
• Hemocytometer
• Centrifuge
• Mouse Erythrocyte Lysing Kit (Catalog # WL2000)
• MagCellect Magnet (Catalog # MAG997) or equivalent
• 12 x 75 mm (5 mL) polystyrene round-bottom tubes
• 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 Mouse Regulatory T Cells

PBMC Preparation

Prepare a single cell suspension of mononuclear cells.

Wash the cells once with excess PBS.

Centrifuge the cells for 10 minutes at 200 x g.

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.

Perform a cell count.

Adjust the cell concentration to at least 20 x 10⁷ cells/mL with cold 1X MagCellect Plus Buffer.

Transfer 20 x 10⁷ cells (1 mL) into a 5 mL polystyrene tube.

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

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

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

Mix gently.

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

Add 1.55 mL of MagCellect Plus Buffer.

Mix gently.

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 naïve CD4⁺ T cells into a new 5 mL tube.

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 naïve CD4⁺ T cells.

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 20 x 10⁷ cells/mL; add 10 μL of the antibody cocktail per 1 x 10⁷ cells being processed; add 10 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.