Confluent rat SMC monolayers were exposed to arterial levels of fluid shear stress (14 dynes/cm2)14 for 24 h in a parallel-plate flow chamber. In brief, flow over the cell culture was obtained by a hydrostatic gradient created through differences in height position between two cell culture media reservoirs, one above and one below the chamber. The fluid level in the upper reservoir was maintained by a roller pump (323E/D, Watson-Marlow Alitea Pump, Wilmington, MA, USA). Shear stress was calculated from the volume flow through the chamber according to the formula (6 x Q x µ)/(b x h2). Q=flow (3 ml/s) µ=viscosity (0.01 dyne second/cm2) b=width of the flow channel (4.4 cm) and h=height of the flow channel (0.054 cm). Flow through the chamber was laminar with an approximate Reynolds number of 7.28 for a mean shear stress level of 14 dynes/cm2. Cells were inspected by phase-contrast microscopy before and after shear stress exposure and static incubation to ensure cell viability and to exclude significant cellular detachment. All experiments were carried out at 37°C in an atmosphere with 100% humidity and 5% CO2.
Growth protocol
Rat aortic SMCs of passage 3-5 were expanded in F-12 HAM/10% fetal calf serum (FCS) containing ascorbic acid (AA) and penicillin-streptomycin (PEST). Prior to all experiments, cells were synchronized for 24 h in F-12 HAM/0.1% bovine serum albumin (BSA) supplemented with AA and PEST.
Extracted molecule
total RNA
Extraction protocol
Cell cultures were scraped from culture dishes, suspended in sterile phosphate-buffered saline (PBS) and transferred to centrifuge tubes. Rat carotid arteries were homogenized in Trizol using a Fastprep homogenizing device (Thermo Scientific, Waltham, MA, USA). Cell suspensions were centrifuged for 5 mins at 900 rpm, the supernatant removed, and the pellets resuspended in 1 ml of Trizol (Qiagen, Hilden, Germany). 200 µl phenol-chloroform was added to the suspensions, and RNA isolation proceeded according to protocol supplied by the manufacturer. Total RNA was eluted in 50 µl RNAse-free water, and RNA quality determined on a RNA Nano chip with an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA) and RNA quantity was assessed using a Nanodrop 1000 spectrophotometer (Thermo Scientific).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix one-cycle protocol from total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Standard protocol, Affymetrix Core center, BEA, Karolinska Institute, Sweden
Scan protocol
Standard protocol, Affymetrix Core center, BEA, Karolinska Institute, Sweden
