The CD56+CD8+/CD8– NK Cell Isolation Kit was developed for the isolation of CD56+CD8+ and CD56+CD8– NK cells from PBMCs by sequential sorting.
CD56+CD8+ NK cells have been shown to have stronger cytotoxic function compared to their CD8– counterparts.
NK cells can be divided into several subsets according to functional and phenotypic differences. CD56+CD8+ NK cells represent approximately 3%, CD56+CD8– NK cells about 4% of all PBMCs in healthy donors. Both NK cell populations differ in the presence of homodimeric CD8 formed by two α-chains. Expression of CD8α/α on NK cells appears to be of significance as the CD56+CD8+ subset was shown to have a stronger cytotoxic function compared to its CD8– counterpart.1
Detailed separation procedure
The isolation of both NK cell subsets is performed in a two-step procedure. First, non-NK cells are labeled with a cocktail of biotin-conjugated antibodies and Anti-Biotin MicroBeads und subsequently depleted by separation over a MACS® Column, which is placed in the magnetic field of a MACS Separator. In the second step, the pre-enriched cells are labeled with CD8 MicroBeads and separated over a second column.
Either the non-labeled CD56+CD8– NK cells or the labeled CD56+CD8+ NK cells can be isolated, using an LD Column or an MS Column, respectively. For isolation of both subsets from one sample, the flow-through of the MS Column is further separated over an LD Column.
Cells isolated using the CD56+CD8+/CD8– NK Isolation Kit can be used, for example, for phenotypical and functional characterization of NK cell subsets, for the analysis of receptor expression, cytokine secretion, or cell-cell interaction with other cells of the innate and adaptive immune system. Isolated NK cells are also used to study the role of NK cells in diseases, e.g., hepatitis infection or cancer, as well as during pregnancy.
For the first magnetic separation (depletion): LS or autoMACS® Columns. For the following positive selection of CD56+CD8+ NK cells: MS or autoMACS Columns. For the isolation of CD56+CD8– NK cells: LD or autoMACS Columns.
Both the CD56+CD8+ and CD56+CD8– NK cell subsets were isolated from human PBMCs using the CD56+CD8+/CD8– NK Cell Isolation Kit. Non-NK cells were depleted using an LS Column. The enriched NK cells were magnetically labeled with CD8 MicroBeads, and CD56+CD8+ NK cells were isolated by positive selection using an MS Column. For isolation of the CD56+CD8– NK cells, the wash and the flow-through fraction of the MS Column were further separated using an LD Column and collected in the flow-through. Cells were fluorescently stained with CD8-PE (clone 3G8) and CD56-APC.
PBMCs before separation
Pre-enriched NK cells after depletion of non-NK cells
Isolated CD56+CD8+ NK cells
Isolated CD56+CD8– NK cells