Pan T Cell Isolation Kit II (human) from Miltenyi Biotec

The Pan T Cell Isolation Kit II is suitable for indirect isolation of T cells (CD3+CD8+ cytotoxic T cells and CD3+CD4+ T helper cells) from human peripheral blood mononuclear cells (PBMCs). All non-T cells, including B cells, NK cells, dendritic cells, granulocytes, erythroid cells and monocytes are depleted by magnetic separation. Unlabeled T cells are present in the column effluent and therefore remain untouched. In a first step, PBMCs are mixed with a cocktail of biotin-conjugated monoclonal antibodies directed against CD14, CD16, CD19, CD36, CD56, CD123 and Glycophorin A. After the addition of anti-biotin MicroBeads, cells are separated using Miltenyi’s MS, LS or XS columns (depending on total cell numbers) in the magnetic field of a suitable Miltenyi Separator. Indirectly magnetically labelled non-T cells remain on the column while untouched T cells can easily be eluted with buffer. The kit includes enough Pan T Cell Biotin-Antibody cocktail (1 ml) and Anti-Biotin MicroBeads (2 ml) for about 10⁹PBMCs and up to 100 separations. The kit can be stored at 4-8°C for at least 7 months without loss of function.

We routinely use Miltenyi’s Pan T cell Isolation Kit II for indirect selection of T cells from human PBMCs suspensions, either directly from fresh preparations, or after stimulation with different antigens. We always receive cell purities of at least 95% as determined by fluorescence activated cell sorting, which is excellent for our most important downstream application, RNA isolation. When we employ fresh PBMC preparations, the overall T-cell yield after separation is about 30% of the number of PBMCs used for this purification. As verified by flow cytometry, this yield is roughly consistent with the CD3+ T cell fraction of PBMCs. The protocol is straight forward and easy to handle. Magnetic labelling including all washing steps will only take about 45 to 55 minutes and magnetic separation with MS and LS columns will take another 10 to 15 minutes. One important advantage is the fact that T cells remain untouched during the whole procedure, making downstream application such as studies on signal transduction or regulation of cytokine expression possible. Moreover the non-T cell fraction, which remains on the column, can easily be eluted outside the magnetic field. Hence, the kit can also be used to deplete T cells from PBMCs. Unfortunately, the Pan T cell Isolation Kit II cannot be used alone. Columns, separation units and a multi stand have to be purchased separately.