MSCs were treated with DMEM-Medium (including 20% FCS, PenStrep and Gentamycin). Starting with a confluency of 50% 200.000 cells per well are seeded and the following experimental procedures are applied for 24h: no treatment (control), rhythmic shear stretch (CMS), exposure to 40% oxygen alone (Hox40), exposure to 80% oxygen alone (Hox80), combination of shear stretch and oxygen 40% (CMS+Hox40), combination of shear stretch and oxygen 80% (CMS+Hox80).
Growth protocol
Pulmonary MSCs are isolated from tracheal aspirates of preterm infants and taken into culture (passage 2-6). For the functional studies 200.000 cells per well are seeded and treated for 72h (treatment group RNA 24h, Group 1-8, see below).
Extracted molecule
total RNA
Extraction protocol
peqGOLD Total RNA Kit
Label
Cy3
Label protocol
Purified total RNA was amplified using the Agilent Low-Input QuickAmp Kit (Agilent, Waldbronn, Germany).
Hybridization protocol
Hybridization was performed following the Agilent protocol at 65°C over-night in an in-situ hybridization oven with slides mounted in Agilent hybridization chambers. Slides were rotated with 2 rpm.
Scan protocol
Slides were scanned using the InnoScan 900 scanner (Innopsys, Carbonne France) at 2 µm/pixel resolution and analyzed with Mapix 8.2.7
Data processing
Raw data was further processed using R (http://www.R-project.org) and limma (Ritchie, M.E., Phipson, B., Wu, D., Hu, Y., Law, C.W., Shi, W., and Smyth, G.K. (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies. NAR 43(7), e47). Background correction was performed with the normexp model using negative control spots and and offset of 1. Spot intensities were quantile normalized between arrays.
