X vivo 20 medium

BM was harvested from wild-type or CtsZ^−/− β-actin-GFP mice by flushing the femurs and tibias with X-VIVO 20 medium (Cambrex) under sterile conditions. The flushed cells were resuspended in PBS, and 1 × 10⁶ nucleated cells were injected into the tail veins of 4-wk-old wild-type or CtsZ^−/− RT2 animals, which were lethally irradiated (950 rads) 6–8 h prior to injection. After 4 wk, recipient mice were bled from the orbital sinus to evaluate BM transplantation efficiency by determining the percentage of GFP⁺ cells using flow cytometry (80.6% GFP⁺ cells for wild-type; 83.7% GFP⁺ cells for CtsZ-deficient animals). Mice were subsequently aged to 13.5 wk for calculation of tumor volume and analysis of the tumor phenotype.

For generation of mo-DCs, monocytes were purified by plastic adherence of PBMCs. For this purpose, cells were seeded (1 × 10⁸/10 ml) into 75 cm² cell culture flasks (Corning, Cambridge, MA, USA) in serum-free X-VIVO 20 medium (Cambrex Bio Science, Verviers, Belgium). After 2 h of incubation at 37 °C/5% CO₂, non-adherent cells were removed. The monocytes were cultured in 10 ml RP10 medium (RPMI-1640 with glutamax-I), supplemented with 10% inactivated foetal calf serum and antibiotics (Invitrogen, Karlsruhe, Germany). GM-CSF (100 ng ml⁻¹; Leukine Liquid Sargramostim; Sanofi, Bridgewater, NJ, USA) and IL-4 (20 ng ml⁻¹; R&D Systems, Wiesbaden, Germany) were added every second day for 7 days. After culture, cells were trypsinized and the reaction was blocked with culture medium. After washing two times with FACS buffer, cells were stained with allophycocyanin-conjugated anti-CD209 Ab (DCN46; BD Biosciences, Heidelberg, Germany) and the indicated workshop Abs using the protocol described above. CD209 was used as the key marker to define mo-DCs.^{24 (link)}