6 protocols
array scanning protocol
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.
nucleic acid hybridization to array protocol
600ng of labelled RNA was hybridised for 16 hours to Agilent SurePrint G3 Human 8x60K microarray Slides.
nucleic acid labeling protocol
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.
nucleic acid extraction protocol
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.).
treatment protocol
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).
growth protocol
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.