cytokine expression (TNF, IL12 p40, IL6 and IFN-a). Similarly, LPS could not change baseline levels of MAP kinases family, but caused a slight decrease in IkBa [51]. The reason why DC cells from SOCS2 knock out animals seem to respond differently from human cells is unclear. The fact that mature DCs in mice instead of immature DCs in humans were treated with LPS might explain the absence of LPS reactivity in DCs from SOCS2 knock out mice. Alternatively, it may exists a functional difference between human and mouse DCs regarding SOCS2. In conclusion, our results indicate that SOCS2 positively regulates maturation of human DCs in vitro. This is achieved by promotion of TLR4 signaling through both the MyD88- dependent and -independent signaling pathway. Materials and Methods Ethics Statement The procedure of human PBMCs isolation was performed following approval by the Ethics Committee of the Karolinska Institute and Karolinska University Hospital. Cell culture media and reagents L-glutamine, penicillin, streptomycin and FCS (Hyclone, Logan, UT); Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden); RPMI-1640 cell culture medium and LPS derived from Escherchia coli O26: B6 (Sigma-Aldrich, St Louis, USA); Recombinant human GM-CSF and recombinant human IL-4 (Invitrogen Biosource, Camarillo, CA); and CD14+ Human monocyte isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) were purchased from the sources indicated. Generation of human monocyte-derived DCs Human PBMCs were isolated from fresh heparinized blood buffy coats (not older than 8 hours) from healthy donors provided from the Department of Transfusion Medicine, Karolinska University Hospital, by a standard procedure of Ficoll-Paque density gradient centrifugation. CD14+ cells were negatively selected by MACS, according to the manufacturer’s instructions. The mean purity of the obtained CD14+ cells was more than 95%, as revealed by flow cytometry. CD14+ cells were subsequently cultured in cell culture flasks (106cells/ml) and in complete RPMI1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100U penicillin and 100U streptomy- cin/ml, 50 ng/ml GM-CSF and 20 ng/ml IL-4 for 6 days. The cells were fed with fresh medium (half original medium volume) containing 100 ng/ml GM-CSF and 40 ng/ml IL-4 on days 2 and 4. Mature DCs were obtained from immature DCs (56105/ml), cultivated for 6 days as described above, by stimulation with 200 ng/ml LPS for 24 h after a first incubation for 2 h in fresh supplemented RPMI 1640 medium with 50 ng/ml GM-CSF and 20 ng/ml IL-4. Transfection Transfection of immature DCs (iDCs) was performed using a commercial kit and a nucleofector machine (Amaxa Co., Ko¨ln, Germany). According to the manufacturer’s instruction, the iDCs were collected on day 6. 107 cells were resuspended in 100 ml human DC nucleofection solution. Small interfering RNA (siRNA) (Qiagen, sequences in Table S1, The control siRNA and the SOCS2 siRNAs were tested in control experiments for effects on mRNA expression of IFNb and IFNa1 measured by Real-time PCR. In a time course study we could not observe any effect on IFNa1 and IFNb, whereas a robust induction was seen by exposing cells to Poly I:C. IFNb expression was slightly increased SOCS2 and DC Maturation by the transfection method however this increase was marginal compared to the effect of LPS on IFNb expression.) was added, and the mixed samples were transferred into certified cuvettes and transfected by using program U002. 500 ml pre-warmed RPMI 1640 medium, supplemented with 10% FCS, L-glutamine, penicillin and streptomycin, was added to each cuvette after transfection. The transfection efficiency of siRNA was measured with Alexa Fluor 488 labeled negative control and Alexa Fluor 488 labeled SOCS2 siRNA and FACS, and it is shown to be near 100% in our experiments (data no shown). The transfected cells were collected and seeded into wells of 6-well plates containing supplemented RPMI 1640 medium with 50 ng/ml GM-CSF and 20 ng/ml IL-4. After 24 hours, the cells were washed and divided into several dishes for stimulation and analysis. Real-time PCR Total RNA was extracted from 1236106 cells using TRIzol (Invitrogen). RNA (1 mg) was treated with DNase I (Promega, Madison, WI) and then reverse-transcribed with a cDNA synthesis kit (Bio-Rad, Hercules, CA). The synthesized cDNA was used as template in a real-time PCR mix according to the manufacturer’s standard protocol (iQ SYBR Green supermix reagents and iQ5 detection system from Bio-Rad, Hercules, CA). The reactions were performed in 20 ml with 2 ml of respective cDNA sample. As a control for the specificity of the real-time PCR a sample without template was included. For each studied gene, a relative standard curve was constructed by serial dilutions using a pool of all cDNA samples. All the measurements were performed in duplicate or triplicates for each sample and normalized to the housekeeping genes RP-II. All primer sequences are provided in Table S2. Flow cytometry Antibodies for staining of human DCs were CD14 (FITC; M5E2), HLA-DR (PerCP; L243), CD 40 (APC; 5C3), CD 80 (PE; L307.4), CD 83 (PE; HB15), CD 86 (APC; B70) (BD Pharmingen, NJ, USA) and CD1a (Pacific blue; HI149) (Bioscience, San Diego, CA). Approximately 56105 DCs were harvested and stained with antibodies for 30 minutes according to standard protocols. After the incubation, the cells were washed with FACS buffer and fixed in diluted FACS lysing solution 10 minutes and finally suspended in 500 ml of FACS buffer. For multiple colors marker staining, the cells were incubated with the combination of FITC-conjugated CD14, Pacific Blue-conjugated CD1a, PE-conjugated CD83 and APC-conjugated CD86, alternatively with APC-conjugated CD40, PE-conjugated CD80 and PerCP-conjugated HLA-DR. Thirty thousand cells were acquired and analyzed on a FACS Arial flow cytometer (BD Biosciences, Heidelberg, Germany). An appropriate gate was set on the basis of scatter properties for excluding dead cells, and only cells within this gate were analyzed. Cells exhibiting a higher mean fluorescence intensity value than cells stained with respective isotype control were considered positive. Nuclear and cytoplasmic extraction and whole-cell lysates iDCs were resuspended in complete RPMI 1640 medium and treated with LPS for different periods. After washing once with cold PBS, nuclear and cytoplasmic protein were extracted from 56106 DCs using a commercial nuclear and cytoplasmic protein extraction kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s instruction. To obtain whole protein lysates, cells were incubated with radio immune precipitation assay buffer (containing 100 ml/ml of a protease inhibitor cocktail solution PLoS ONE | www.plosone.org 6 September 2009 | Volume 4 | Issue 9 | e7178