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| Status |
Public on Oct 10, 2022 |
| Title |
EyeBank_cultured_hCECs_monoculture_Exos_hCECs_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Primary human corneal epithelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
exosomes: hCECs exosomes
age: 71
|
| Treatment protocol |
800-100 ug of exosomes were added to cell cultures for a incubation of 48h before harvesting cells. Vehicle alone (HBS) was used for controls.
|
| Growth protocol |
To isolate hCECs and hCFs, post-mortem corneas were incubated with dispase in HEPES buffer overnight at 4 °C to separate the epithelium from the stroma. The stroma was then cut into small pieces and incubated with collagenase H until the ECM was fully digested by the enzyme. hCECs were treated with trypsin for 15 min at 37 °C to separate the cells from each other. hCECs were grown in DH medium (Dulbecco–Vogt modification of Eagle's medium with Ham's F12 in a 3:1 ratio supplemented with 5% FetalClone II serum, 5 μg/mL of insulin, 0.4 μg/mL of hydrocortisone, 10 ng/mL of epidermal growth factor, 0.212 mg/mL of isoproterenol hydrochloride, antibiotics (100 IU/mL of penicillin, and 25 μg/mL of gentamycin) on lethally irradiated human fibroblasts feeder layers (iHFL) until they reached confluence. hCFs were grown in DME medium (Dulbecco–Vogt modification of Eagle's medium supplemented with 10% fetal calf serum and antibiotics). All cells were grown under 8% CO2 at 37°C and culture medium was changed every 2 or 3 days. To isolate hCEnCs, the Descemet membrane was carefully peeled off and incubated overnight in culture medium at 37°C. It was then digested with EDTA 0.02% buffered solution for 1 hour. Cells were detached by pipetting up and down with a flamed-polished pipette. hCEnCs were then seeded on fibronectin/collagen (FNC)-coated plastic culture dishes and grown until they reached confluence in a proliferation medium (Opti-MEM-I medium supplemented with 8% fetal bovine serum, 5 ng/ml epidermal growth factor, 0.08% chondroitin sulfate, 20 μg/mL ascorbic acid and 100 IU/mL of penicillin and 100 μg/mL of streptomycin). The culture medium was then replaced with a maturation medium (Opti-MEM-I medium supplemented with 8% fetal bovine serum and penicillin/streptomycin) when hCEnCs reached confluence, and were grown further for an additional 7 to 28 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Cat#74104)
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA (hCECs monoculture) or 100 ng RNA (hCECs from hTECs) using the One-Color LowInput QuickAmp Labeling Kit One-Color (Agilent) according to the manufacturer's instructions
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| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >8.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole SurePrint G3 Human GE 8x60K (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScanner (G4900DA) using one color scan setting for 1x60k array slides (Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of hCECs cultivated in monoculture until they reached near-confluence with hCECs exosomes
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10,7 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
Data were analyzed using the ArrayStar V4.1 (DNASTAR, Madison, WI) software. All data generated from the array were also analyzed by RMA (Robust Multiarray Analysis) for background correction of the raw values. They were then transformed in log2 base and quantile normalized before a linear model was fitted to the normalized data in order to obtain an expression measure for each probe set.
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| |
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| Submission date |
Oct 09, 2022 |
| Last update date |
Oct 10, 2022 |
| Contact name |
Gaëtan Le-Bel |
| E-mail(s) |
gaetan.lebel17@gmail.com
|
| Organization name |
CUO-Recherche
|
| Lab |
Sylvain Guérin
|
| Street address |
Local H2-00, Hôpital du Saint-Sacrement,1050 Chemin Sainte-Foy
|
| City |
Québec |
| State/province |
Quebec |
| ZIP/Postal code |
G1S 4L8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE215102 |
Gene expression in human corneal epithelial cells (hCECs), human corneal fibroblasts (hCFs) or human corneal endothelial cells (hCEnCs) with or without exosomes derived from hCECs, hCFs or hCEnCs |
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