|
| Status |
Public on Mar 22, 2019 |
| Title |
H-1303_P5_4w_SCB 2 |
| Sample type |
RNA |
| |
|
| Source name |
H-1303, passage 5, cryopreserved with STEM-CELLBANKER for 4 weeks
|
| Organism |
Homo sapiens |
| Characteristics |
cell characters: normal chondrocytes
cell provider: Articular Engineering
cell line name: Human Chondrocytes, Cryopreserved, Primary, CDD-H-2610
cell lot: H-1303
passage number: 5
cryoprotectant medium: STEM-CELLBANKER
cryopreservation period: 4 weeks
|
| Treatment protocol |
Each viable cells were suspended in either STEM-CELLBANKER (ZENOAQ), STEM-CELLBANKER DMSO Free (ZENOAQ), or 10% DMSO (SIGMA, D2650) in 20% Fetal Bovine Serum, MSC-Qualified (FBS) (Invitrogen) containing culture medium. The cell suspension was dispensed into a cryopreservation tube (Corning, 430488) and was frozen in a BICELL freezing vessel (Nippon freezer) at -80℃ for overnight, and was transferred into a liquid nitrogen tank or -150°C refrigerator for more than 4 weeks or 1 year. The frozen cells were quickly thaw in a 37°C water bath and were plated into culture vessel. When the cell confluency becomes 60–90 %, the cells were subcultured. The cell confluency becomes 70–90 % again, then the cells were harvested for DNA microarray analysis.
|
| Growth protocol |
ADSCs were maintained in MesenPRO RS Medium (Invitrogen), according to the manufacturer’s instructions. UC-MSCs were maintained using Mesenchymal Stem Cell Growth Kit for Adipose and Umbilical-derived MSCs - Low Serum (ATCC, PCS-500-040) plus Mesenchymal Stem Cell Basal Medium for Adipose, Umbilical and Bone Marrow-derived MSCs (ATCC, PCS-500-030), according to the manufacturer’s instructions. Yub621c and Yub625 were cultured in 1x Penicillin-Streptomycin (gibco)-containing MesenPRO RS Medium (Invitrogen), according to the manufacturer’s instructions. NHAC-kn and H-1303 were maintained using CGM Chondrocyte Growth Medium BulletKit (Lonza, CC-3216) and Chondrocyte ReagentPack Subculture Reagents (Lonza, CC-3233), according to the manufacturer’s instructions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using ISOGEN (Nippon Gene) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 150 ng of total RNA using Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA (specific activity >6.0 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, 55 ul of 2x HiRPM Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8x60K Microarray Kit (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 by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides.
|
| Description |
Gene expression in human cell
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software GX13.1 (Agilent) using default parameters.
|
| |
|
| Submission date |
Mar 22, 2016 |
| Last update date |
Mar 22, 2019 |
| Contact name |
Yuzuru Ito |
| Organization name |
National Institute of Advanced Industrial Science and Technology (AIST)
|
| Department |
Biotechnology Research Institute for Drug Discovery
|
| Street address |
Central 6, Higashi 1-1-1
|
| City |
Tsukuba |
| State/province |
Ibaraki |
| ZIP/Postal code |
3058566 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE79496 |
DMSO-free chemically defined cryopreservation of human mesenchymal stem cells and chondrocytes |
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