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

This protocol provides a basic guide for the magnetic selection of CD4⁺ T lymphocytes from preparations of peripheral blood mononuclear cells (PBMC) or splenocytes.

In this protocol, a mononuclear cell suspension is first 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. The enriched cell preparation is then available for a variety of applications including tissue culture, immune status monitoring, and flow cytometry.

See Details

Why Isolate CD4⁺ T Cells?

CD4⁺ T cells develop in the thymus and differentiate into various subsets of more specialized T lymphocytes. These cells differentiate into subsets of specialized effector T lymphocytes in response to distinct environmental cues and the activation of specific transcription factors. The isolation of CD4⁺ T cells is an important step in the preparation of multiple subsets including Th1, Th2, Th17, Th9, Th22, and regulatory T cells. CD4⁺ T cell subsets secrete characteristic combinations of cytokines which enables them to exert diverse functions including the recruitment and activation of additional immune cells, the dampening of ongoing immune responses, and the maintenance of immunologic memory.

Preparation of PMBC and Splenocytes

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

Isolation of CD4⁺ T Cells from PBMC or Splenocytes

MagCellect™ CD4⁺ T cell isolation kits are designed to isolate CD4⁺ T cells by negative selection. The resulting cell preparations are highly enriched with CD4⁺ T cells with no detectable CD8⁺ T cells. MagCellect kits are available for the isolation of CD4⁺ T cells (human or mouse), naïve CD4⁺ T cells (human or mouse), and memory CD4⁺ T cells (human).These kits contain sufficient quantities of the following reagents to process 1 x 10⁹ total cells:

• Biotinylated antibody cocktail to remove cells other than CD4⁺ T cells
• 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

CD4⁺ T Cell Selection Procedure

See Details

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.

1. 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.

 

 

2. 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 1 x 10⁸ cells/mL.

 

 

3. 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.

 

 

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

 

 

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

 

 

6. 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.

 

 

7. Recovery of desired 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.

 

 

8. 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 desired enriched CD4⁺ T cells. The cells are now ready for counting and further downstream applications.

View Larger Image

Enrichment of CD4+ T Cells from Peripheral Blood Mononuclear Cells Using the MagCellect Human CD4+ T Cell Isolation Kit. Ficolled human PBMC before (A) and after (B) isolation of CD4+ T cells using the MagCellect Human CD4+ T Cell Isolation Kit (Catalog # MAGH102) were stained with PE-conjugated Mouse Anti-Human CD3 epsilon Monoclonal Antibody (Catalog # FAB100P) and FITC-conjugated Mouse Anti-Human CD4 Monoclonal Antibody (Catalog # FAB3791F).

View Larger Image

Enrichment of Naïve CD4+ T Cells from PBMC Using the MagCellect Human Naïve CD4+ T Cell Isolation Kit. Ficolled human PBMCs before (A) and after (B) processing with the MagCellect Human Naïve CD4+ T Cell Isolation Kit (Catalog # MAGH115). Cells were stained with an APC-conjugated Mouse Anti-Human CD4 Monoclonal Antibody (Catalog # FAB3791A) and PE-conjugated Mouse Anti-Human CD45RA Monoclonal Antibody (Catalog # FAB1430P).

• 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.