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E-GEOD-23618 - Transcriptome profiling of dendritic cell subtypes

Status
Submitted on 12 August 2010, released on 27 October 2010, last updated on 15 May 2013
Organism
Homo sapiens
Samples (12)
Array (1)
Protocols (7)
Description
In this study transcriptome profiling of dendritic cell subtypes was performed using various human dendritic cells. Monocyte-derived DC: CD14+ monocytes were obtained from buffy coats by Ficoll - gradient centrifugation and immunomagnetic cell separation using anti-CD14-conjugated microbeads. Monocytes were resuspended into 6-well culture dishes at a density of 1.5 x 10 million cells/ml and cultured in RPMI 1640 supplemented with 10% FBS containing 800 U/ml GM-CSF and 500 U/ml IL-4. Cells were cultured for 5 days and the IL-4 and GM-CSF addition was repeated at day 2. Dermal DC (DDC) and Langerhans Cells (LC) from skin: Human skin specimens were obtained from healthy donors undergoing corrective breast or abdominal plastic surgery after informed consent. Thick slices (3 mm) of skin containing the epidermis and the dermis were cut by use of a dermatome. Slices of skin were cut in pieces of 1 cm2 and incubated with Dispase II for 30–60 min at 37°C. The epidermis and dermis were separated with tweezers and washed with PBS. To isolate LC, the epidermal sheets were incubated with PBS with 0.05% trypsin for 10 min at 37°C, and a single-cell suspension was prepared by pushing the tissue through 100 µm pore nylon cell strainers with a plunger of a 2-ml syringe. Epidermal cell suspension was enriched for LC by density centrifugation over Lymphoprep and CD1a-guided MACS (Miltenyi Biotec). To isolate DDC, the dermis was incubated with PBS containing Dispase and Collagenase A at 37°C for 2 h, after which, a single-cell suspension was prepared by pushing through 100 µm pore nylon cell strainers with a plunger of a 2-ml syringe. Cell suspension was enriched for DDC by CD1a-guided MACS. Blood Cd1c+ DC: Blood CD1c+ DC were isolated from periferal blood. BDCA1+ (CD1c) DCs were incubated with a lineage-specific PE-labeled Ab mixture, HLA-DR-allophycocyanin, and BDCA1-FITC. Lineage-negative and HLA-DRhigh DCs, positive for BDCA1 were sorted on a FACS and collected in tubes containing 1 ml of 100% FBS. A total of 40,000–100,000 cells was isolated (>98% pure). Dendritic cell subtypes were differentiated in vitro from monocytes and primary dendritic cells were isolated from human skin and blood.
Experiment type
transcription profiling by array 
Contacts
Istvan Szatmari <szatmari@indi.biochem.dote.hu>, LAJOS SZELES <szelesl@dote.hu>, Lajos Szeles, Laszlo Nagy, Malin Lindstedt, Saskia Santegoets
Citation
Research resource: transcriptome profiling of genes regulated by RXR and its permissive and nonpermissive partners in differentiating monocyte-derived dendritic cells. Széles L, Póliska S, Nagy G, Szatmari I, Szanto A, Pap A, Lindstedt M, Santegoets SJ, Rühl R, Dezsö B, Nagy L.
MIAME
PlatformsProtocolsVariablesProcessedRaw
Files
Investigation descriptionE-GEOD-23618.idf.txt
Sample and data relationshipE-GEOD-23618.sdrf.txt
Raw data (1)E-GEOD-23618.raw.1.zip
Processed data (1)E-GEOD-23618.processed.1.zip
Array designA-AFFY-44.adf.txt
R ExpressionSetE-GEOD-23618.eSet.r
Links