Embryos used for fibroblast isolation were obtained from a time mated CD1 mouse. At 19-days post conception, the pregnant mouse was euthanized by CO2 asphyxiation and immediately swabbed with 70% ethanol in a sterile hood. Tendon, corneal and skin fibroblasts were isolated according to Spector et al {Spector, Goldman Robert D, et al. 1998 381 /id}. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% FCS in a 5% CO2 humidified atmosphere at 37 °C, and were subcultured when they reached approximately 80% confluence. Cells were cultured until five population doublings, at which point they were seeded onto a gelatin-coated (1% w/v) glass plate (7cm x 10cm) and allowed to adhere for approximately seven hours prior to stimulation. STIMULATION: Fluid flow was applied to cells using a parallel plate flow chamber described previously {Ohtsuka, Ando, et al. 1993 341 /id}. Wall shear stress (tw) was calculated according to the equation tw = 6mQ/(bh2), where m is the fluid viscosity (0.001 Pa s), Q is the fluid flow rate (ml/s), and b and h are the width (5.5 cm) and height (0.04 cm) of the flow channel, respectively. The assembled system was maintained at 37 °C in a 5% CO2 humidified incubator. In this study, tendon, corneal and skin fibroblasts were subjected to a shear stress of 0.1 dynes/cm2 for 14 hours, with a flow perfusate of DMEM supplemented with 2% FCS and 1% penicillin/streptomycin.
Extracted molecule
total RNA
Extraction protocol
RNA extractions were carried out with the Absolutely RNA RT-PCR Miniprep kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. Contaminating RNases were inactivated in solutions upon treatment with 0.1% (v/v) DEPC, while RNaseZap was used according to the manufacturer's instructions to eliminate RNase contamination on work surfaces and pipettes.
Label
biotin
Label protocol
Standard Affymetrix GeneChip Eukaryotic Two-Cycle Target Labeling
Hybridization protocol
standard Affymetrix procedures
Scan protocol
standard Affymetrix procedures
Description
Primary fibroblast from the cornea of the fetal mouse, mechanically stimulated by fluid flow
Data processing
Robust Multichip Average (RMA, module Affy 1.4.32 of Bioconductor)
