Splenic DC Isolation and Gene Expression

As previously described (Stanley et al., 2008 (link)) mice were infected with L. donovani and sacrificed 5 hours later. Spleens were digested in collagenase type IV (1 mg/ml; Worthington, Lakewood, NJ) and deoxyribonuclease I (0.5 mg/ml; Worthington) at RT for 45 minutes. Splenocytes were processed as described above. Splenic DCs were isolated from splenocyte preparations by positive selection with anti-CD11c MACS beads, according to the manufacturer’s instructions (Miltenyi Biotec). DCs were also stained for MHCII and CD86 and analyzed by flow cytometry, as described above. Purified DCs were stored in buffer RLT and homogenized in QIAshredder columns (both from QIAGEN). Total RNA was extracted from purified DC using the RNeasy Mini Kit with on-column DNase digestion (both from QIAGEN). RNA samples were reverse transcribed into cDNA using the cDNA Archive Kit (Applied Biosystems, Foster City, CA). RT-qPCR for Il12 and Il10 was performed on CF384 Touch Real-Time PRC Detection System (BIO-RAD) using the TaqMan Gene Expression Assay (Applied Bioscience). Relative quantification was performed using the comparative C_T method relative to Hprt and beta2m.