Isolation of dendritic cells from mouse lymph nodes

Lymph nodes are the primary sites of T-cell stimulation by dendritic cells (DC). After contact with antigens, DCs migrate to draining lymph nodes from the skin and other tissues (1-3). Investigation of the morphology and function of lymph node DCs may provide important information about the role of these cells in normal and pathological conditions. Therefore, lymph nodes are popular sites for the isolation of dendritic cells. Dendritic cells isolated from lymph nodes represent interdigitating" DCs that are localized in T-dependent regions of lymph nodes. DCs represent about 1% of the total population of lymph node cells. Therefore, in order to perform almost any functional tests, the DC fraction should be enriched. The most practical way to enrich the DC fraction is to use a density gradient. Several gradients-metrizamide (4), Nycodenz (5), and Percoll (6)-have successfully been used for enrichment of DCs obtained from different sources. When isolating DC from lymph nodes, density gradient separation produces a population of DC with a purity of 40-50%. Most contaminating cells are lymphocytes with a small fraction (usually less than 5%) of macrophages. The choice of lymph nodes is dependent on the purpose of the experiment. The most commonly used lymph nodes are axillary, inguinal, and popliteal. DCs can be further enriched using monoclonal antibodies and flow cytometric cell sorting, magnetic beads separation, panning, or cytotoxic elimination with complement. All these methods are based on the negative selection of DCs using anti-T, anti-B, and anti-macrophage antibodies. Since the first step of isolation involves gradient centrifugation, granulocyte contamination is negligible and further purification steps do not require use of anti-granulo-cyte antibodies.