|
| Status |
Public on Jan 07, 2014 |
| Title |
CD14_Placenta1_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
CD14
|
| Organism |
Homo sapiens |
| Characteristics |
cd14: YES
time [days of culture]: 0
cell type: CD14
source placenta: 1
biological replicate: 2
|
| Treatment protocol |
MGC (9 days of culture) were compared with initial placental macrophages (CD14+).
|
| Growth protocol |
Placentas from healthy at-term pregnancies and vaginal deliveries were collected in the Gynecology-Obstetrics Department of the Hôpital de la Conception (Marseille, France). Chorionic villi from the maternal interface were cut along their entire length without taking the membrane of the chorionic plate. They were then cut into small pieces of approximately 1 cm² and rinsed in PBS. Placenta samples were digested. Briefly, placenta samples were incubated in a solution consisting of HBSS, MgSO4, DNase I and 2.5% trypsin buffered with HEPES for 45 min and then for 30 min under gentle agitation at 37°C. The digestion products were filtered through 100-µm pores, incubated in 50 ml tubes containing 2 ml FCS and centrifuged at 1,000 × g for 15 min. The cells were counted, deposited on a Ficoll cushion and centrifuged at 700 × g for 20 min. Mononuclear cells were recovered, and the placental macrophages were isolated using magnetic beads coated with anti-CD14 Abs. The purity of the isolated CD14+ cells was verified by flow cytometry and was greater than 97%. Placental MGCs were obtained as follows. CD14+ macrophages (2 × 105 cells per assay) were seeded in 24-well plates containing glass coverslips (day 0) and cultured in DMEM-F12 containing 10% FCS and antibiotics for 9 days. The morphology of the placental macrophages and the percentage of MGCs were determined by May-Grünwald Giemsa staining using the HemaColor kit.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using an RNeasy Mini kit (Qiagen, Courtaboeuf, France). The quantity and quality of total RNA were assessed using the Nanodrop (Thermo Scientific, Illkirch, France) and a 2100 Bioanalyzer (Agilent Technologies, Massy, France). The RNA was eluted in 30 µl of water and stored at -20°C.
|
| Label |
Cy3
|
| Label protocol |
In brief, 400 ng RNA were labeled with cyanine-3-CTP using the Low RNA Input Fluorescent Amplification Kit from Agilent Technologies.
|
| |
|
| Hybridization protocol |
RNA was analyzed using microarray chips including 45,000 probes (4x44K Whole Human Genome, Agilent Technologies) and One-color Microarray Based Gene Expression Analysis. Hybridization was performed at 65°C using the in situ Hybridization Plus kit (Agilent Technologies) for 17 hours.
|
| Scan protocol |
Arrays were scanned with a pixel size of 5 µm with the DNA Microarray Scanner G2505B. Image analysis and correction of intra-array signals were performed with the Feature Extraction Software A.9.1.3 (Agilent Technologies).
|
| Data processing |
Microarray data analysis was performed using the R and the Bioconductor software suite. Raw data were filtered and normalized using the Agi4x44PreProcess library. Unsupervised and supervised analyses were done using hierarchical clustering, correspondence analysis (COA) (made4 library) and Significance Analysis of Microarray (SAM) algorithm (siggenes library). Supervised analysis was performed using SAM. Genes were considered to be differentially expressed when the false discovery rate was below 1% and absolute fold change (FC) was above 4.
|
| |
|
| Submission date |
Jun 15, 2012 |
| Last update date |
Jan 07, 2014 |
| Contact name |
Julien Textoris |
| E-mail(s) |
[email protected]
|
| Phone |
+33 472 119 546
|
| Organization name |
bioMérieux
|
| Department |
Medical Diagnostic Discovery Department (MD3)
|
| Lab |
Joint Research Unit - bioMérieux / HCL
|
| Street address |
Hôpital Edouard herriot - Pavillon P; 5 place d'Arsonval
|
| City |
Lyon |
| ZIP/Postal code |
69437 |
| Country |
France |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE38747 |
Regulatory role of multinucleated giant cells derived from placental CD14+ macrophages: Pathophysiological implications |
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