T-PCR analysis of FcγR expression on pulmonary DC. Purified lung DC were taken up in TriZol® Reagent (Invitrogen, Karlsruhe, Germany), total RNA was isolated from frozen samples with a chlorophorm-propanol-ethanol extraction procedure and cDNA synthesis was carried out via reverse PF-02341066 manufacturer transcriptase (Qiagen, Hilden, Germany). Quantitative real-time RT-PCR analysis was performed with an iCycler® (Biorad, Munich, Germany) and QuantiTect SYBR® Green PCR kit (Qiagen) in order to determine the levels of FcγRI-IV mRNA, normalized to tubulin and using published FcγRI-III primers 33, 34. For detection of FcγRIV transcripts,
the following FcγRIV-specific primers were used:
sense, 5′-CAGAGGGCTCATTGGACA-3′; antisense, 5′-GTGATTTGATGCCACGGT-3′. The PCR condition was 95°C, 15 min one cycle, followed learn more by 94°C, 15 s, 52.5°C, 30 s and 72°C, 30 s for 40 cycles for all primer pairs. DC were isolated from mouse spleen or lungs as previously described 35–37. In brief, the organs were cut into small fragments, digested with collagenase and DNase I (Sigma) and enriched by gradient centrifugation using Nycodenz reagents (Axis-Shield, Oslo, Norway) with a density of 1.073 for lung DC and 1.077 for splenic DC. DC were then enriched by negative depletion using magnetic separation and an antibody cocktail containing anti-Gr1, anti-B220, anti-erythrocytes, anti-CD19 and anti-CD3. To prevent
DC maturation during the isolation protocol, the procedure was carried out on ice, with the exception of the initial 20 min digestion with collagenase/DNase, which was performed at room temperature. This protocol excluded B220+ “plasmacytoid DC” from the DC preparation 38. DC were labeled with CD11c (HL3, FITC or PE), CD4 (GK1.5, FITC or PE), and CD8 (53-6.7, APC) monoclonal antibodies (all BD Biosciences, Heidelberg, Germany). Lung DC were stained for CD11c and MHC class II (2G9), CD11b (M1-70), CD103 (M290) (all BD PharMingen, Germany), CD16 (275005, IgG2a, Alexa 647), CD32 (K9 361, IgG2b, Alexa 647), CD64 (290322, IgG2a, plus goat-anti-rat APC, Invitrogen) (all R&D Systems, Germany) or isotype control antibodies. Analytical and RAS p21 protein activator 1 preparative fluorescent-activated cell sorting was done on a FACSAria (BD Biosciences, San Jose, CA, USA), or a Mo-Flo (Cytomation, Fort Collins, CO, USA) instrument and sorts were usually 95–98% pure. Gating strategy for analysis and sort of lung DC and lung macrophages (CD11c+MHC class IIlow) is shown in Fig. 2B. For spleen-derived DC, dead cells were excluded by DAPI or PI-staining, and CD11c+ cells were gated and analyzed for CD4 and CD8 expression. BMDC were generated by flushing out the BM from tibia and fibula of B6 mice.