donor: E condition: Asthmatic nanoparticle: Air dose: 0 mg/m3 dose description: control air time: 1 hour aerosolization + 24 hours incubation
Treatment protocol
Prior to performing the experiments, the MucilAir™ cells were stabilized in culture for at least one week, while the medium was refreshed every 2–3 days. The basolateral culture medium was refreshed approximately 24 h before exposure. The MucilAir™ cells were rinsed with saline solution (0.9% NaCl,1.25 mM CaCl2 and 10 mM HEPES buffer) approximately 24 h before and again just before exposure to ensure that each individual model contained a mucus layer of comparable thickness. Right before exposure, the inserts were transferred to the exposure device. The stainless-steel wells of the exposure device contained MucilAir culture medium to feed the cell monolayer from the basal side during a 1-hour exposure.
Growth protocol
MucilAir™ fully differentiated bronchial epithelial models (Epithelix Sárl, Geneva, Switzerland), reconstituted from primary human cells of healthy or asthmatic donors, were used. The cells were maintained on 24-well Transwell® culture supports at an air-liquid interface using MucilAir™ culture medium, supplemented with 1% amphotericin and 0.5% gentamicin, in a humidified incubator at 37 °C with 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Cells were stabilized for another 24 h with 0.7 ml MucilAir culture medium at the basal site in a humidified incubator (ca. 37 °C, 5% CO2) after exposures. Total RNA was isolated from cell lysates with RNeasy Plus Mini Kit according to manufacturer's instructions (Qiagen, GmbH, Hilden, Germany). Desalting of the samples was required and thus, additional purification was performed by ethanol precipitation with 3M sodium acetate (Thermo Fisher Scientific Inc., Wilmington, NC, USA)
Label
Cy5
Label protocol
Samples were amplified using T7 RNA polymerase method and the cRNAs were further labeled with Cy3 and Cy5 dye, according to the manufacturer's recommendations.
Hybridization protocol
Labeled cRNAs were hybridized to Agilent 2-color 60-mer oligo arrays according to the manufacturer's recommendations.
Scan protocol
Slides were washed and scanned with Agilent Microarray scanner G2505C, according to the manufacturer's recommendations.
Description
Exposed for 1h to nanoparticle aerosol, incubated for 24h
Data processing
Raw intensity values were obtained from Agilent Feature Extraction software and quality checked according to Agilent standard procedures. The median foreground intensities were imported into R software and analyzed with BioConductor package Limma.
