Isolation of rat intestinal lymph DC.

Dendritic cells (DCs) migrate constitutively from the intestine via the lymph to the mesenteric lymph nodes. These migrating intestinal lymph DCs (ilDCs) carry antigens acquired in the intestine and play important roles in both the initiation of immune responses and the maintenance of oral tolerance. The ilDC population is made up of at least three functionally different DC subsets. Like many DC populations, ilDCs are exquisitely sensitive to their environment, changing their phenotype and maturing in response to the procedures associated with their extraction from solid tissues. We have developed and refined a method for collecting and purifying these DC subsets from rats, without inducing them to mature. This method involves two separate surgical procedures, separated by at least 6 weeks. Initially, mesenteric lymph nodes are removed. After the animals have fully recovered we cannulate the thoracic duct and collect the ilDCs on ice, minutes after they have left the lymph vessel. The DCs are then enriched using magnetic beads and purified by flow cytometric sorting. We describe this method here, including our recent refinements to limit the use of the restraining "Bollman" cage.