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.
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
- Sterile 15 mL conical centrifuge tubes
- Column rack, such as R&D Systems Catalog # RACK