Comment[ArrayExpressAccession]	E-MTAB-1536
MAGE-TAB Version	1.1																					
Investigation Title	Stem cell coculture																					
Comment[Submitted Name]	Stem cell coculture
Experiment Description	This study aims to to compare the gene transcription profiles of endothelial cells and stem cells,  when they are cultured alone or when they are cultured together.  Thus there are two major questions - how do the cells differ, and how do the cells influence each other's gene expression.                              Thus there are 4 types of sample: endothelial cell monoculture, endothelial cell coculture, stem cells monoculture, stem cell coculture.    There are also 4 biological replicates (independent experiments) leading to 16 array data files.																					
Experimental Design	cell_type_comparison_design	in_vitro_design	co-expression_design																			
Comment[AEExperimentType]	transcription profiling by array																					
Comment[AEExperimentDisplayName]	Transcription profiling by array of human mesenchymal stem cells and endothelial cells to investigate down-regulation of cytokine-induced leukocyte recruitment																					
Experimental Factor Name	cell type	phenotype																				
Experimental Factor Type	cell type	phenotype																				
Quality Control Type	biological replicate																					
Quality Control Term Source REF	EFO																					
Public Release Date	2013-10-10																					
Person Last Name	Nash																					
Person First Name	Gerard																					
Person Mid Initials	B																					
Person Email	g.nash@bham.ac.uk																					
Person Phone	4.41214E+11																					
Person Address	The Medical School, Birmingham B15 2TT, UK																					
Person Affiliation	University of Birmingham																					
Person Roles	submitter																					
PubMed ID																						
Publication Author List	N. Thin Luu, Helen M. McGettrick, Christopher D. Buckley, Phil Newsome, G. Ed Rainger, Jon Frampton,  Gerard B. Nash																					
Publication Title	Crosstalk between mesenchymal stem cells and endothelial cells leads to down-regulation of cytokine-induced leukocyte recruitment																					
Publication Status	In preparation																					
Protocol Name	P-MTAB-31455	P-MTAB-31456	P-MTAB-31457	P-MTAB-31458	P-MTAB-31459	P-MTAB-31460
Protocol Type	growth protocol	treatment protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning protocol																
Protocol Description	Isolation and culture of endothelial cells (EC) and mesenchymals stem cells  (MSC)- Human umbilical cords were obtained from the Human Biomaterials Resource Centre (University of Birmingham) after informed consent.  HUVEC were isolated from umbilical cords as previously described and cultured in M199 supplemented with 20% FCS, 10 ng/ml epidermal growth factor, 35 ?g/ml gentamicin, 1 ?g/ml hydrocortisone (all from Sigma-Aldrich, Poole, UK), and 2.5 ?g/ml amphotericin B (Life Technologies; Carlsbad CA). Human bone marrow derived MSC were purchased from Lonza and cultured in the manufacturer?s recommended medium (MSCGMTM Mesenchymal Stem Cell Growth Medium BulletKit, Lonza Ltd, Basel, Switzerland). They were expanded through 3 passages and used uniformaly at that passage.	Coculture of EC with MSC -  MSC and EC were co-cultured on opposite sides of filters (6-well format) with 0.4?m pores.  EC were seeded on the inner surface first and cultured for 24h to form a confluent layer.  Then MSC (5x105cells) were added to the other surface and cocultured with EC for 24h.    Cells were retrieved separately from either side of the filter using trypsin for analysis of gene expression .  For comparison, MSC or EC were cultured individually on identical filters (monocultures).	 mRNA was isolated from EC or MSC retrieved from either side of filters, or from monocultures, using the RNeasy Mini Kit (Qiagen, Crawley, U.K.). 	For microarray analysis, 25ng of each sample of RNA was labelled with Cy3 dye as per protocol in the Low Input Quick Amp Labelling Kit (Agilent Technologies ? 5190-2305). A Specific Activity of greater than 6.0 was confirmed by measurement of 260nm and 550nmwavelengths with a NanoDrop ND-1000 Spectrophotometer. 	600ng of labelled RNA was hybridised for 16 hours to Agilent SurePrint G3 Human 8x60K microarray Slides. 	After hybridisation slides were washed and scanned with a High Resolution C Scanner (Agilent Technologies). Feature extraction was performed using Agilent Feature Extraction Software, with no background substraction. 																
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO	EFO																
Term Source Name	EFO
Term Source File	http://www.ebi.ac.uk/efo																					
SDRF File	E-MTAB-1536.sdrf.txt