CD4+ T Cell Isolation from PBMC or Splenocyte Preparations by T Cell Enrichment Column: R&D Systems

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

In this protocol, a leukocyte suspension is incubated with a mixture of monoclonal antibodies and is then loaded onto the T Cell Subset Column. B cells and non-selected T cell subsets are tagged by the antibody cocktail and bind to the anti-immunogloblin coated glass beads. Monocytes bind to the column resin via interactions with their Fc receptors. The highly enriched cell preparation in the column eluate is then available for a variety of applications including tissue culture, immune status monitoring, and flow cytometry.

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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

CD4⁺ T Cell Enrichment Columns 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. T Cell Enrichment Colulmns are available for the isolation of CD4⁺ T cells (human, mouse, or rat), naïve CD4⁺ T cells (human or mouse), and memory CD4⁺ T cells (human or mouse). Both the human and mouse CD4⁺ T Cell Enrichment Columns are available in Small and Mini sizes for the processing of different quantities of cells. Mini size columns require smaller volumes of buffer as indicated in the following protocol.

T Cell Enrichment Column Kits contain the following reagents:

CD4⁺ T Cell Subset Columns
Monoclonal Antibody Cocktail
Column Buffer Concentrate (10X)

Other Materials Required

Pipettes and sterile pipette tips
Hemocytometer
Sterile 15 mL conical centrifuge tubes
Ficoll-Hypaque™
Column rack, such as R&D Systems Catalog # RACK

Other Equipment Required

Centrifuge
Microscope

CD4⁺ T Cell Enrichment Column Procedure

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Prepare 75 mL of 1X Column Buffer for each column to be used by mixing 7.5 mL of 10X Column Buffer Concentrate with 67.5 mL sterile deionized or distilled water.

Prepare a single cell suspension of human leukocytes by following the Leukocyte Preparation Protocol.

Suspend 2 x 10⁸ cells (1 x 10⁸ cells for Mini columns) in 1-2 mL 1X Buffer and add the contents of 1 vial of Monoclonal Antibody Cocktail (1 mL; 0.5 mL for Mini columns). Gently mix the cell-antibody suspension and incubate at room temperature for 15 minutes.

While the cells are incubating with the antibody cocktail, begin washing the column with 1X Buffer as described in Step 6.

Wash the cells twice with 10 mL 1X Buffer by spinning the cells at 300 x g for 10 minutes and decanting the supernatant after each wash. Suspend the washed cell pellet in 2 mL of 1X Buffer.

Place the column in a rack or ring stand. Remove the top cap of the column first to avoid drawing air into the bottom of the column. Then remove the bottom cap. Allow the column fluid to drain into a waste receptacle. Rinse the outside tip of the column with a 70% ethanol solution during this time to ensure sterile cell processing.

Wash the column with 10 mL (6 mL for Mini columns) 1X Buffer and collect the eluate in a waste receptacle. The column is now ready for the cells.

Replace the waste receptacle with a sterile 15 mL conical centrifuge tube.

Load the cell-antibody suspension onto the column and allow it to enter into the column. The suspension will displace some of the buffer in the column which can be collected.

Incubate the column containing the cell suspension at room temperature for 10 minutes.

After the incubation step, elute the cells by adding 12 mL (8 mL for Mini columns) 1X Buffer to the top of the column. Collect the eluate until it appears clear.

Centrifuge the collected cells at 250 x g for 5 minutes. Decant the supernantant and resuspend the cells in the appropriate buffer or culture medium. The enriched CD4⁺ T cells are now ready for counting and further downstream applications.

Technical Hints

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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.
In order to best determine column performance, we advise users to retain a small portion of the starting cell population. Following cell enrichment with the column, it is then be possible to perform immunophenotyping analysis on both starting and eluted cells. This information when combined with the actual number of cells loaded and recovered can then be used to calculate the percentage recovery of the target cell population.
Some of the salts in the 10X Column Buffer Concentrate may precipitate after storage at 2 - 8 °C. Should this be the case, do not carry out the 1:10 buffer dilution (as indicated in Step 1) until all salts are in solution. This may be achieved by warming the 10X Column Buffer Concentrate bottle in a 37° C water bath for 5 - 10 mintues. Once there is no longer evidence of precipitates, the 10X Column Buffer Concentrate may be diluted 1:10 to prepare the 1X Column Buffer.