Aging Aging

302 Grubeck-Loebenstein, Saurwein-Teissl, and Romani3. DC differentiation: Monocytes/monocyte-enriched fractions are cultured at a densityof about 3–5 × 10 5 /mL in GM-CSF (800 U/mL) + IL-4 (1000 U/mL) for6–9 d. The cells have then acquired the characteristics of DCs of intermediatematurity. Cell markers of mature DCs are expressed, but at low density. Furtherdifferentiation/maturation can be achieved by a 2–3-d exposure to either monocyte-conditionedmedium (see Subheading 2.3.), or, alternatively, a cocktail(TNFα ±IL-1 ± IL-6 + prostaglandin E 2 ) that mimics monocyte-conditionedmedium (54,55) in the continued presence of GM-CSF ± IL-4. When TGF-β isadded together with GM-CSF and IL-4 from the beginning of the cultures, LCdevelopment is most pronounced (20).4. Feeding of the cultures: The cultures have to be fed every other day, starting ond2 of culture. To this end 1 mL of culture medium is carefully aspirated from thewells. To compensate for evaporation it is replaced by 1.5 mL of fresh culturemedium containing 1600 U/mL of GM-CSF and 1000 U/mL of IL-4.3.5. Generation of DCs from Human Blood CD34 + Progenitors1. PBMCs are obtained from buffy coat by standard Ficoll–Hypaque/Lymphoprepdensity centrifugation, washed twice, and centrifuged through 10% BSA toremove platelets.2. Purification of CD34 + cells: 5 × 10 8 cells are incubated for 5 min at 4°C in 500 µLof FcR blocking reagent (included in the MiniMACS multisort kit). Five hundredmicoliters of CD34 Multisort microbeads are then added and the cells left toincubate at 4°C for 60 min. After this incubation period cells are centrifuged andresuspended in PBS-EDTA. Microbead-labeled cells (10 8 cells) are loaded ontoeach column of the kit in 500 µL and inserted into the magnetic field. Columnsare washed 3× and then removed from the magnetic field. CD34 + cells bound tobeads are cosequently eluted with 1.5 mL buffer and then loaded onto a furthercolumn. The procedure is repeated. Finally, the microbead-labeled CD34 + cellpopulation is incubated with Multisort Release Reagent for 10 min at 4°C torelease the beads from the cells and loaded onto another column in a magneticfield. The eluate which contains the unbound CD34 + cells is centrifuged through10% BSA in PBS for 10 min at 600g and the pellet is resuspended. Purity controlby FACS analysis (see Note 13).3. Differentiation of DCs from stem cells: CD34 + cells are put into 24-well cultureplates (1–10 5 cells/well) in culture medium and stimulated every 2 d withGM-CSF (400 U/mL) and TNF-α (500 U/mL). A greater yield will be achievedwhen flt-3 (100 ng/mL), SCF (20 ng/mL), and TGF-β (0.5 ng/mL) are additionallypresent. The latter cytokine works particularly well under serum-free conditions,under which it prevents apoptosis (21).4. Harvesting of DCs: DCs are best harvested after 7–14 d of culture.3.6. Generation of DCsfrom Murine Bone Marrow CD34 + Progenitors1. Preparation of bone marrow: Remove muscles from the femurs and tibias.Immerse the bones in a Petri dish with 70% ethanol for 1 min, wash twice in