The Purification of Large Numbers of Antigen Presenting Dendritic Cells from Mouse Spleen

Dendritic cells (DC) are found at low frequency in lymphoid and non-lymphoid tissues. Different DC subsets are adept at different roles in immunity in diverse scenarios of attack by infectious agents, as well as in the maintenance of self-tolerance. A key element in the ability of DC to initiate adaptive immune responses is their capacity to capture and process antigen, whether from pathogens, vaccines or self-components, and present it to T cells. Our typical procedure for isolation of the different DC types from murine spleen involves their digestion from the tissue using collagenase, selection of cells of light density, and negative selection for DC. DC may then be separated into their functionally distinct subpopulations using immunofluorescent labeling and flow cytometric cell sorting. If the availability of mice is limiting, our protocol can cater for DC numbers boosted by the administration of fms-like tyrosine kinase 3 ligand (Flt3L), directly via subcutaneous injection or via the introduction of a Flt3L secreting melanoma cell line. Large numbers of in vitro equivalents of the spleen DC subsets may also be produced by culturing bone marrow with Flt3L. If flow cytometric sorting time is a limitation splenic DC subpopulations may instead be separated using a combination of fluorescent antibody labeling and anti-fluorochrome magnetic beads. Careful segregation of these functionally distinct subpopulations of DC will enable a thorough examination of their antigen processing and presenting capabilities.