|
| Status |
Public on Apr 29, 2019 |
| Title |
cultured bovine granulosa cells untreated rep 4 |
| Sample type |
RNA |
| |
|
| Source name |
cultured bovine granulosa cells, untreated
|
| Organism |
Bos taurus |
| Characteristics |
cell type: cultured bovine granulosa cells
treatment: untreated
|
| Treatment protocol |
Bovine ovaries were obtained from a local abattoir. Antral granulosa cells were aspirated from small to medium sized follicles (<6mm), collected in phosphate buffered saline (PBS, pH 7.4) and isolated by centrifugation from the buffer. Living cells were counted using the trypan blue exclusion method and cryo-preserved in freezing media (fetal calf serum containing 10 % DMSO; Roth, Karlsruhe, Germany). For seeding, the GC were thawed and immediately transferred into α-MEM and centrifuged at 500 x g for 3 min to ensure fast removing of the freezing media. Afterwards the cell pellet was diluted in α-MEM containing L-Glutamin (2 mM), sodium bicarbonate (0.084%), BSA (0.1%), HEPES (20 mM), sodium selenite (4 ng/ml), transferrin (5 µg/ml), insulin (10 ng/ml), non-essential amino acids (1 mM), penicillin (100 IU/ml) and streptomycin (0.1 mg/ml), FSH (20 ng/ml; Sigma Aldrich, Steinheim, Germany), R3 IGF-1 (50 ng/ml; Sigma Aldrich), and androstenedione (2 µM; Sigma Aldrich). Improved attachment of plated GC was achieved with Collagen R coated wells (0.02%; Serva, Heidelberg, Germany). Cells were cultured at a density of 1.0x10E5 living cells per well. Cells were additionally treated with sodium L-lactate (30 mM; Sigma Aldrich), sodium chloride as vehicle control (30 mM; Sigma Aldrich) or left untreated. If not stated otherwise all reagents were purchased from Merck Millipore (Berlin, Germany). GC were cultured for 8 days at 37 °C and 5% CO2 and two-third of the media with or without L-lactate or vehicle was exchanged every other day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using the NucleoSpin RNA Kit including removal of genomic DNA with RNAse free rDNAse (Macherey-Nagel).
|
| Label |
Biotin
|
| Label protocol |
Biotinylated RNA were prepared according to the standard Affymetrix protocol (GeneChip® Expression 3’Amplification One-Cycle Target Labeling and Control Reagents).
|
| |
|
| Hybridization protocol |
The RNA was hybridized overnight (45°C) in the GeneChipR Hybridization Oven (Affymetrix) to the Affymetrix Bovine Gene 1.0 ST arrays. Washing and staining were performed according to the Affymetrix standard protocol.
|
| Scan protocol |
The scanning of the microarray was done with the GeneChip Scanner 3000 HR (Affymetrix) and image processing was done with the software AGCC (Affymetrix).
|
| Data processing |
Raw data were processed with the Expression Console (V1.4.1.46; Affymetrix), where normalization, background reduction and a gene-level summary was done using the RMA method (Robust Multichip Average).
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| |
|
| Submission date |
Oct 17, 2018 |
| Last update date |
Apr 29, 2019 |
| Contact name |
Jens Vanselow |
| E-mail(s) |
vanselow@fbn-dummerstorf.de
|
| Phone |
+49 38208 68750
|
| Organization name |
Leibniz Institute for Farm Animal Biology
|
| Department |
Reproductive Biology
|
| Lab |
Experimental Reproductive Biology
|
| Street address |
Wilhelm-Stahl-Allee 2
|
| City |
Dummerstorf |
| ZIP/Postal code |
18196 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16500 |
| Series (1) |
| GSE121408 |
L-lactate induces specific genome wide alterations of gene expression in cultured bovine granulosa cells |
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