infection: Human Cytomegalovirus Strain TB40E mock cell type: Dendritic Cells
Treatment protocol
Human foreskin fibroblasts (HFF) were isolated from foreskins of male neonates by trypsin treatment and were used for experiments between passages 10 and 25. Alternatively, MRC-5 fibroblasts were purchased from Sigma-Aldrich, Taufkirchen, Germany. Fibroblasts were cultured in MEM containing glutamine (2.4 mmol/l), gentamicin (100 g/ml), and fetal calf serum (5 %). For high-titer virus preparations, fibroblasts were infected at a low multiplicity of infection (MOI) with TB40E. Six days after infection, 100 ml cell-free supernatant was concentrated by ultracentrifugation at 80,000 × g for 70 min, and pelleted virus particles were resuspended in appropriate volumes of RPMI. 1 x 106 DCs were incubated with the virus preparation at 37 °C (MOI=50) in complete RPMI medium supplemented with IL-4 and GM-CSF. After 2h, cells were washed once with Hanks and resolved in fresh medium. For mock-infection, cells were exposed to fresh medium as an appropriate control for cell-free virus concentrates. TB40E-infected or mock-infected DCs were collected after 24 h to assess the efficiency of infection. Cells were washed in PBS / 2% fetal calf serum (FCS) / 2mmol EDTA, cytocentrifuged for 5 min at 550 rpm and fixed on glass slides with acetone for 10 min. HCMV immediate early (IE) antigen expression was detected by indirect immunofluorescence staining. Slides were incubated with monoclonal antibody directed against the non-structural proteins IE72 and IE86 (pUL122/123, Biosoft, Paris, France) at the appropriate dilution for 90 min at 37°C, then were incubated with Cy3-conjugated Fab´2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, USA) for 45 min at 37°C. Nuclei were counterstained with DAPI. The slides were washed in PBS for 5 min after each incubation step. The percentage of infected cells was determined by immunofluorescence microscopy. As previously reported by Grigoleit et al., 2002, IE antigen was detected in 35% – 75% of the DCs infected with TB40E.
Extracted molecule
total RNA
Extraction protocol
Peripheral blood mononuclear cells (PBMCs) were isolated from 50-ml Buffy Coat blood (Blutspendedienst Tübingen / Frankfurt, Germany) by Ficoll-Hypaque density gradient centrifugation. CD14+ cells were isolated from PBMCs by magnetic-associated cell sorting (MACS) using paramagnetic microbeads conjugated to monoclonal anti-human CD14 antibodies (Miltenyi, Bergisch Gladbach, Germany). Monocytes were seeded into six-well plates (2,5 x 106 cells / 3 ml / well) in RPMI-1640 (+ GlutaMAX) medium containing 10% fetal calf serum (FCS) and 100 mg/ml gentamicin sulfate (Refobacin® 80 mg, Merck, Darmstadt, Germany), supplemented with 100 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; Leukomax, Novartis, Basel, Switzerland) and 20 ng/ml Interleukin-4 (IL-4; R&D Systems, Wiesbaden, Germany). Cultures were fed with fresh medium and cytokines every other day. DC differentiation was monitored by light microscopy and purity of DC culture was analyzed after 7 days by flow cytometry (FACSCalibur, Becton Dickinson, Heidelberg, Germany). At least 95% of the DC population stained was positive for CD1a, CD40, CD80 and CD86 and was negative for CD14 and CD83. Standard qiagen Isolation protocol
Label
biotin
Label protocol
Standard Affymetrix Labeling protocol
Hybridization protocol
Standard Affymetrix Hybridisation protocol
Scan protocol
Standard Affymetrix Scanning protocol
Description
RMA normalized data not provided by submitter
Data processing
RMA normalisation, ArrayAssist DAtaanalysis with t-test