|
| Status |
Public on Dec 05, 2006 |
| Title |
Human lymphatic endothelial cells TNFalpha_Rep 3 |
| Sample type |
RNA |
| |
|
| Source name |
Primary lymphatic endothelial cells, cultured in vitro with TNFalpha, derived from skin samples obtained from healthy human donors during elective surgery.
|
| Organism |
Homo sapiens |
| Characteristics |
Human dermal microvascular lymphatic endothelial cells (HDLEC) were prepared from healthy female adults undergoing elective surgery (breast reduction and abdominoplasty).
|
| Biomaterial provider |
Human samples obtained from Radcliffe Infirmary Hospital_Oxford (with full ethical approval)
|
| Treatment protocol |
Human skin biopsy was digested overnight (4ºC) with Dispase®, 2 mg/ml (Gibco) in PBS, pH 7.5, to remove the epidermis. Cells were released from human tissue by scraping, passed through a 70μm cell strainer (BD Falcon) and expanded in 0.1% gelatine-coated flasks in complete EGM-2 MV medium. HDLEC were lifted with Accutase (PAALaboratories) then immuno-selected with mouse anti-human LYVE-1 mAb, followed by magnetic retrieval with appropriate MACS bead preparations (Miltenyi Biotech). HDLEC were cultured for 48h in EGM2MV medium supplemented with TNFalpha, 1 ng/ml.
|
| Growth protocol |
HDLEC were cultured in complete EGM-2 MV medium(Cambrex Bio Science Walkersville Inc.) supplemened with 1ng/ml TNFalpha at 37ºC, 5 % CO2 in a humidified atmosphere, using plastic tissue culture flasks that had been pre-coated with gelatin (0.1%, Gibco)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen RNeasy.
|
| Label |
Biotin
|
| Label protocol |
8 microgram total RNA used in Affymetrix one cycle target labeling kit cat. no. 900493.
|
| |
|
| Hybridization protocol |
Fragmented labeled cRNA (15µg) was used in 300µl hybridization cocktail containing spiked controls (Affymetrix #900299). A 200µl of this hybridization cocktail was used on the chip and incubated at 45°C for 16 h in the hybridization oven, rotating at 60 rpm. Following hybridization, the arrays were processed using a Genechip Fluidics Station 400 according to recommended protocols (EukGE-WS2v5, Affymetrix) of double-staining and post-hybridizations washes.
|
| Scan protocol |
Fluorescent images were captured using gene Array Scanner 3000 and GCOS1.2 software (Affymetrix).
|
| Description |
Here we have addressed important question of inflammation-induced mechanism for leukocyte transmigration of lymphatic vessel endothelium by comparing global gene expression profile of normal dermal lymphatic endothelial cells with/without TNFa added to the culture.
|
| Data processing |
We have used the genechip robust multi-array average expression measurements (GC-RMA) as implemented in BioConductor R statistics (www.bioconductor.org). Cell Intensity files (CEL) were used to obtain expression values for all the 54,675 probe sets (transcripts) on each array.
|
| |
|
| Submission date |
Nov 08, 2006 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Dilair Baban |
| E-mail(s) |
dilair.baban@well.ox.ac.uk
|
| Phone |
+44(0)1865287521
|
| Organization name |
University of Oxford
|
| Department |
Wellcome Trust Centre Human Genetics
|
| Lab |
Genomics
|
| Street address |
Roosevelt Drive
|
| City |
Oxford |
| ZIP/Postal code |
OX3 7BN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE6257 |
An inflammation-induced mechanism for leukocyte transmigration of lymphatic vessel endothelium. |
|
| Relations |
| Reanalyzed by |
GSE49910 |
| Reanalyzed by |
GSE64985 |
| Reanalyzed by |
GSE119087 |
