|
| Status |
Public on Jul 05, 2024 |
| Title |
NHK_DP_4h |
| Sample type |
RNA |
| |
|
| Source name |
Keratinocytes with double paracrine stimulation for 4h
|
| Organism |
Homo sapiens |
| Characteristics |
group: Double paracrine stimulation
stimulus: Fibroplast conditioned medium
timepoint: 4h
tissue: Skin
gender: Male
cell type: Normal Human Epithelial Keratinocytes
array id: 6235821016
|
| Treatment protocol |
KSFM medium was added to a culture of NHK and collected after 12h to stimulate sub-confluence monolayers of HDF for 18 hours. This supernatant from the conditioned HDF culture was used to stimulate keratinocytes (NHK_DP_Xh) for the specified time in a double paracrine manner. NHK_DP_C0/4/12 samples are used as controls, wherein NHK are stimulated with KSFM medium for 12 h, then again for 18h and then again for the specified time. NHK_Xh are keratinocyte mono-cultures sampled after the specified time. After each conditioning the collected media were centrifuged (200 xg/ 5 min) and then used to stimulate the respected other cells. Time and volume was considered to have adequate soluble factors content and sufficient medium nutrients.
|
| Growth protocol |
Normal human keratinocytes (NHK) and human dermal fibroblasts (HDF) were derived from foreskin as previously described (König & Bruckner-Tuderman, 1994; Stark et al, 1999). NHK were cultivated in keratinocyte serum free medium (KSFM, Invitrogen, Carlsbad, CA, USA) supplemented with recombinant human EGF (rhEGF) and pituitary bovine extract (BPE). Cells were kept under a humidified environment with 5% CO2 and 37oC. Keratinocytes were used up to passage 4-5 and density of 4-9 x 104 cells/cm2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Trizol reagent, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit. Quality control was performed with Agilent Bioanalyser.
|
| Label |
Cy3
|
| Label protocol |
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
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| |
|
| Hybridization protocol |
Hybridization was performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 100 ng cRNA/µl, unsealed in a wet chamber for 20h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes. After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals were developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays were dried and scanned.
|
| Scan protocol |
Microarray scanning was done using a Beadstation array scanner, setting adjusted to a scaling factor of 1 and PMT settings at 430. Data extraction was done for all beads individually
|
| Data processing |
All arrays were normalized together using the quantile normalization algorithm with background correction.
Quantile normalization based on idat file information and BGX file annotation using Limma in R
|
| |
|
| Submission date |
Jul 04, 2024 |
| Last update date |
Jul 05, 2024 |
| Contact name |
Hauke Busch |
| E-mail(s) |
hauke.busch@uni-luebeck.de
|
| Phone |
+49-451-3101-8470
|
| Organization name |
University of Lübeck
|
| Department |
Lübeck Institute of Experimental Dermatology
|
| Street address |
Ratzeburger Allee 160
|
| City |
Lübeck |
| State/province |
Schleswig-Holstein |
| ZIP/Postal code |
23538 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10558 |
| Series (1) |
| GSE271501 |
Cell-Cell communication between skinkeratinocytes and fibroblats is non-redundantly coded through inflammatory cytokines |
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