Aging Aging

Dendritic Cells in Old Age 303Ca 2+ - and Mg 2+ -free PBS, and put them into a Petri dish with RPMI-1640. Cutoff both ends (epiphyses) of each bone with scissors. Make a marrow suspensionby flushing out the shafts with a syringe containing about 2 mL of RPMI-1640per bone. Larger clumps are removed by passage through nylon mesh. Red bloodcells are depleted by hypotonic lysis with NH 4 Cl. The marrow is then depleted ofmature leukocytes by treatment with antibodies for B cells, T cells, granulocytes,and complement. For the exact procedure consult Subheading 3.1. (see Note 14).2. Culturing the cells: Bring the cells to 5–6 × 10 5 /mL in culture medium containingGM-CSF (final concentration of 200 U/mL) and plate 1–1.5-mL volumes into thewells of a 24-well plate.3. Propagation and differentiation of DCs: The wells are washed and fed every 2 d.Early in the course of the culture (d 2–4) one rinses off the many nonadherentgranulocytes that are developing. On d 4 the rinsing step with RPMI-1640 isomitted. By d 4 one begins to see aggregates of growing DCs attached to theadherent stroma. The aggregates are round, in contrast to macrophage colonies,which are flattened and dispersed. One can also recognize many cell processes(“veils”) extending from the periphery of the aggregates, giving them a “hairy”appearance. By d 6, the wells are usually covered with many aggregates (“balls”)of proliferating DCs. TNF-α (500 U/mL) when added during the last 2 d of culturesignificantly increases the percentage and yield of mature DCs (MHC class II + ;CD86 + ).4. Harvesting of DCs: Between d 6 and 8, dislodge the growing aggregates from theadherent stroma and transfer them to tissue culture dishes. Cells are pooled,centrifuged, and plated in 10 mL of fresh culture medium containing GM-CSFper 100 mm Petri dish at a maximal cell number of 10 × 10 6 per dish. During the24 h following this transfer, free, nonproliferating, mature DCs are released fromthe aggregates. They can be collected by gently swirling the cells from the plate.Harvesting the mature progeny is best performed after 8 or 9 d of culture (seeNote 15).3.7. Generation of DCs from Human Bone MarrowSufficient amounts of bone marrow for the generation of DCs will only rarelybe available from healthy aged person. Upon suspension culture in GM-CSF +TNF-α + SCF for 12–14 d an effective yield of about 1.7 × 10 6 mature DCs persingle milliliter of adult human bone marrow can be obtained. The generationof DCs from bone marrow CD34 + cells has yet to be optimized with respect to (1)increasing the low percentage of DCs in the bulk cultures and (2) avoidance of FCS.4. Notes4.1. Purification of DCs (LCs) from Murine Skin1. Cell yields: Depending on the mouse strain (56), 3–5 × 10 6 primary epidermalcells, containing 1.4–3% LCs, can be obtained per mouse. Lower yields have tobe expected in aged animals (25).