Abstract
This method allows you to isolate different subclass mononuclear cells, like B-cells, T cells, CD4+ and CD8+ T, from mouse spleen. By conjugating cells with specific antibodies and subsequently magnetic beads isolation, using the technique from Miltenyi, this allows a high purity.
Keywords: T cell, Purification, Lymphocyte, Isolation
Materials and Reagents
Equipment
Procedure
B-cells, total T cells and/or single positive CD4+ and CD8+ T cells, were purified from spleen cells by magnetic separation with the Mini-MACS system [Miltenyi, 1990] (http://www.miltenyibiotec.com). The scheme illustrates how to manage the procedure. Figure 1. Schematic view of the experimental strategy using magnetic MACS beads to isolate CD4+ cells. Cells were incubated with CD4-,CD8-,CD90- and CD43-antibody conjugated magnetic beads. The cell suspensions were subjected to column selection and placed in the magnetic separator. Flow-through was discarded. Then the column was washed with separation puffer to increase purity and removed afterwards from the magnetic separator. With a plunger the magnetically labelled cells were flushed out of the column.
Recipes
Acknowledgments
The authors thank the German Research Foundation (DFG) and the University Erlangen-Nuremberg for funding. We thank A. von Berg and L. Sologub for excellent technical assistance.
References
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