|
| Status |
Public on Jun 11, 2012 |
| Title |
Macrophage_from_iPSC+NLRP3wt |
| Sample type |
RNA |
| |
|
| Source name |
iPSC
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: iPS cells
|
| Treatment protocol |
Undifferentiated human ESC and iPSC lines were cultured on mitotically-inactivated SNL feeder cells with Primate ES cell medium supplemented with 4 ng/ml bFGF. During the differentiation of the cells into macrophages, cells were cultured under 37°C , with 5% CO2 and 5% O2. On day 0, the iPSCs were plated at a ratio of 1:15 onto a mitotically-inactivated OP9 feeder layer on 100 mm cell culture plates in αMEM (Invitrogen) containing 10% FBS and 1% Antibiotic-Antimycotic (Invitrogen) supplemented with 50 ng/ml VEGFα (R&D Systems). On day 5, the medium was changed. On day 10, the differentiating iPSCs were collected by trypsinization, and Tra-1-85+ CD34+ and KDR+ hematopoietic progenitors were sorted on a FACS Aria II instrument (BD Biosciences). The progenitors were plated at 2 x 104 cells on another mitotically inactivated OP9 feeder layer on 100 mm cell culture plates or at 3 x 103 cells/well in 6 well cell culture plates in αMEM containing 10% FBS and 1% Antibiotic-Antimycotic supplemented with 50 ng/ml IL-3, 50 ng/ml SCF, 10 ng/ml TPO, 50 ng/ml FL and 50 ng/ml M-CSF (all R&D Systems). On day 18, the medium was changed. On day 26, differentiating cells were collected with Accumax (Innovative Cell Technologies), and CD14+ iPS-MPs were purified on an autoMACSpro instrument (Miltenyi Biotec).
|
| Growth protocol |
We expanded the fibroblasts in DMEM (Nacalai tesque) containing 10% FBS (Invitrogen) and 0.5% penicillin and streptomycin (Invitrogen). To generate iPS cells, we introduced OCT3/4, SOX2, KLF4 and c-MYC by using ecotropic retroviral transduction into fibroblasts expressing the mouse Slc7a1 gene. Six days after transduction, the cells were harvested and re-plated onto mitotically inactivated SNL feeder cells. The next day, we replaced the medium with Primate ES cell medium (ReproCELL) supplemented with 4 ng/ml bFGF (Wako). Three weeks after this period, individual colonies were isolated and expanded. Cell culture was performed under 37°C , with 5% CO2 and 21% O2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was column-purified with the RNeasy kit (Qiagen) and treated with RNase-free DNase (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
20 µg of total RNA was first amplified with WT-OvationTM Pico System (Nugen). The RNA was labelled with Cy3 using Genomic DNA Enzymatic Labeling Kit (Agilent Technologies).
|
| |
|
| Hybridization protocol |
Labelled cDNA was fragmented with Gene Expression Hybridization Kit (Agilent Technologies), followed by hybridization at 65˚C for 17 hrs.
|
| Scan protocol |
Images were acquired by DNA Microarray Scanner (Agilent Technologies)
|
| Description |
iPS cells carrying wild type NLRP3 derived from fibroblasts (FIBRO), clone 1
|
| Data processing |
Raw data were processed with FeatureExtraction version 10.7. Background signal was subtracted.
|
| |
|
| Submission date |
Jun 10, 2012 |
| Last update date |
Jun 11, 2012 |
| Contact name |
Akira Watanabe |
| E-mail(s) |
a.watanabe@aw-lab.kyoto
|
| Organization name |
Kyoto University
|
| Department |
Medical Innovation Center
|
| Street address |
Shogoin-Kawahara-cho 53, Sakyo-ku
|
| City |
Kyoto |
| State/province |
Kyoto |
| ZIP/Postal code |
606-8507 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE38626 |
Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic mosaicism and drug discovery |
|
