CD8+ cytotoxic T lymphocytes are the main effector cells of cell-mediated immunity; they also play a critical role in the elimination of cells infected by pathogens and in tumor regression. Isolation of a highly pure population of CD8+ T cells is vital for different experimental and clinical applications.
Magnetic-activated cell sorting (MACS) is currently accepted as a standard method for cell separation. MACS technology utilizes tiny super-paramagnetic microbeads for labeling targets; separation is achieved by using columns in which magnetically-labeled cells are retained and unlabeled cells can pass through.
The CD8+ T Cell Isolation Kit II from Miltenyi Biotec is an indirect magnetic labeling system for the isolation of untouched human CD8+ T cells from peripheral blood mononuclear cells by depletion of non-CD8+ T cells (negative selection). Non-CD8+ T cells are incubated with a cocktail of biotin-conjugated antibodies against CD4, CD14, CD16, CD19, CD36, CD56, CD123, TCR gamma/delta and CD235a (Glycophorin A) in order to deplete CD4+ T cells, gamma/delta T cells, B cells, NK cells, monocytes, dendritic cells, granulocytes and erythroid cells. Anti-biotin microbeads are then added to the antibody-tagged cells without any washing steps. The magnetically labeled non-CD8+ T cells can be depleted by holding them on MS or LS columns in the magnetic field of a MACS separator, and unlabeled CD8+ T cells are collected as they pass through the columns. I usually use an autoMACS separator; the 'Deplete' program is recommended. Miltenyi Biotec also offers a similar kit for isolating untouched human CD4+ T cells where CD8+ T cells are depleted instead of CD4+ T cells.
This kit is recommended when untouched CD8+ T cells are required where direct labeling of these cells might interfere with downstream applications. Isolation of CD8+ T cells using this kit is easy, but it is important to evaluate the purity of these cells following their isolation. This kit is sufficient for 109 total cells (up to 100 separations of 107 cells). It is very common to lose a certain amount of your reagents, as the shelf life of this type of kit is approximately six months. Therefore, if a small number of separations are usually performed, reagents are likely to become expired before they are completely used. It would be great if the shelf life of such kits could be increased, especially since they are expensive and these isolations are performed for research and not routine purposes.