Description
Background information
CD8+ DCs coexpress CD11c, CD205, MHC class II, CD40, CD80, CD86, and are negative for CD4 and CD11b. In spleen and lymph nodes, CD8+ DCs are found at moderate levels (23% and 17% of all CD11c+ DCs, respectively) and are located in the T cell areas. Skin-draining lymph nodes harbor an additional CD8+ DC subset (CD4-CD8+CD11b+CD205+) which accounts for 33% of all CD11c+ DCs in this organ and is thought to represent the mature form of Langerhans cells.
Detailed separation procedure
The isolation of CD8+ DCs is performed in two steps. First, DCs are enriched by depletion of T cells, NK cells, and B cells with a cocktail of biotin-conjugated antibodies and Anti-Biotin MicroBeads. Subsequently, CD8+ DCs are positively selected from the enriched DC fraction with CD8a MicroBeads.
Downstream applications
CD8+ DCs isolated using the CD8+ Dendritic Cell Isolation Kit are suitable to analyze antigen uptake, antigen processing, T cell activation or tolerance induction, cross-priming of cytotoxic T cells, and T cell polarization by CD8+ DCs. Isolated splenic CD8+ DCs have been analyzed for the expression of a signal regulatory protein, which plays a role in the migration of murine Langerhans cells.1They additionally have been isolated and adoptively transferred to study their role in the induction of regulatory CD4+ T cells.2 The CD8+ Dendritic Cell Isolation Kit was also used to analyze the expression and regulation of EBV-Induced Gene 3 (EBI3) in mouse DC subsets.3
Columns
For the first magnetic separation (depletion): LD or autoMACS® Columns. For the second magnetic separation (positive selection): MS or autoMACS Columns.
Overview
The CD8+ Dendritic Cell Isolation Kit is an indirect magnetic labeling system for the positive selection of CD8+ DCs from single-cell suspensions of spleen and lymph node.
