cell type: Dendrític Cells infection: control time: 24hr
Treatment protocol
Ten mdDCs cultures were mock-infected or infected with DENV1/60, DENV1/435 or DENV1/480 at an MOI of 5 for two hours in the absence of FBS. The viral inoculum was removed and cells were washed and cultured in RPMI 1640 plus 10% of FBS and 100 IU/µg/mL of penicillin/streptomycin (Gibco-BRL, Grand Island, NY).
Growth protocol
Monocyte-derived dendritic cells (mdDCs) were generated using peripheral blood from healthy volunteers after informed consent and approval of the FIOCRUZ Research Ethics Committee (number 514/09). Mononuclear cells were separated by density gradient using Lymphocyte Separation Medium (Lonza, Walkersville, USA) and CD14+ cells were purified by magnetic immunosorting (Miltenyi Biotec, Auburn, CA), according to the manufacturer’s recommendations. CD14+ cells were cultured for 6-7 days in RPMI 1640 medium containing 100 ng/mL interleukin-4 (IL-4) and 50 ng/mL granulocyte-monocyte colony stimulating factor (GM-CSF; PeproTech, Rocky Hill, NJ), 10% fetal bovine serum (FBS) and 100 IU/µg/mL of penicillin/streptomycin and 2 mM of L-Glutamine (Gibco-BRL, Grand Island, NY). The cultures were assessed by flow cytometry and only cultures with more than 80% of CD1a+ and less than 5% of CD14+ purity were used. C6/36 cells were grown in Leibovitz-15 medium supplemented with 5% FBS and 0.26% of tryptose and 25 µg/mL of gentamicin (all reagents from Gibco-BRL, Grand Island, NY).
Extracted molecule
total RNA
Extraction protocol
Cells were collected at 8, 24 and 48 hours post infection (hpi) and used to extract total RNA using RNaesy Mini Kit according to the manufacturer’s recommendations (QIAGEN, Valencia, CA). The RNA of all ten mdDC cultures was pooled (75 µg of each) for microarray experiments and compared to mock infected cells using Human Gene 1.0 st v. 1 array GeneChip slides from Affymetrix (Santa Clara, CA). The RNA pool was processed as according to the manufacturer’s recommendations (Affymetrix, Santa Clara, CA) for the amplification and in vitro transcription, purification, tagging, hybridization and scanning of the slides (GeneChip® Scanner 3000).
Label
biotin
Label protocol
Single-stranded cDNA was generated from the amplified cRNA with the WT cDNA Synthesis Kit (Affymetrix) and then fragmented and labeled with the WT Terminal Labeling Kit (Affymetrix).
Hybridization protocol
Samples were hybridized with GeneChip Human Gene 1.0 ST Arrays (Affymetrix) and scanned at the LNLS- Campinas Brazil facility.
Scan protocol
Array scanning was performed according to the manufacturer's instruction (Affymetrix)
Data processing
RMA expression value derived from Expression Console software; core-gene analysis The generated files were analyzed for quality by the software Expression Console (Affymetrix, Santa Clara, CA). The same software was used to normalize the data by the RMA method. Log-ratio between DENV1/60, DENV1 435, or DENV1 480 infected-mdDCs and mock-infected-mdDCs intensity signal values were generated and a list of differential expressed genes was obtained by using a 2 our higher fold-change. Data clustering was performed by the Cluster 3.0 software with a Euclidian’s distance. Tool for Analysis of GO Enrichments (TANGO), in Expander software was used to obtain a GO with statistical enhancer values of 0.05, and, NCBIEntrez Gene database (www.ncbi.nih.gov) and the Gene Ontology database (www.geneontology.org), to define the most relevant cell signaling and metabolic pathways during mdDC infection with DENV1 60, DENV1 435, or DENV1 480.
