BEAS-2B cells were cultured in 6-well Transwells with BEGM Medium. After 24h, basal medium was changed, apical medium was removed and cells were kept in air-liquid interface for 24h, before exposure. For combined expousre, cells were exposed to diesel CAST model aerosol for 2hours (or clean air), in an vitro exposure system. After that, basal medium was changed and cells were placed in a cell culture incubator for 24 hours. After that, all cells were exposed to 4 mg of whole birch pollen dispersing it with 0.5 bar pressured air into the Pollen Sedimentation Chamber, for different incubation times. Cells were kept all the time at 37°C and 5% CO2. For the pollen exposure alone, the same amount of birch pollen was used (4mg) and exposure was performed as explained above.
RNA was obtained with the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany), with additional 1% ß-Mercaptoethanol (Roth, Karlsruhe, Germany), according to the manufacturer’s protocol. The quantification and quality of the RNA was assessed with an UV-vis spectrophotometer (NanoPhotometer® N60, IMPLEN, Munich, Germany) and the Agilent 2100 Bioanalyzer RNA Nano chip (Agilent Technologies, Waldbronn, Germany), respectively.
RNA samples (input of 100ng) were spiked (One-Color RNA Spike-in Kit, Agilent Technologies, Waldbronn, Germany), Cy3-labelled (Low Input Quick Amp Labeling Kit, one-color, Agilent Technologies, Waldbronn, Germany) and purified on RNeasy mini spin columns (QIAGEN). Generated Cy3-labelled cRNA was quantified with NanoPhotometer® N60, IMPLEN, Munich, Germany.
The Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes with Agilent fragmentation mix, according to the manufacturers instructions. After that, 25uL of 2x Agilent hybridization buffer was added to the fragmentation mixture and 40uL of the samples was hybridized to One-Color SurePrint G3 8x60K Human gene expression arrays (Agilent Technologies, Inc, Waldbronn, Germany) for 17 hours at 65°C in a rotating Agilent hybridization oven. After incubation time, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1, at 500rpm and 1 minute with 32°C GE Wash buffer 2 (Agilent), at 500rpm. Slide was placed in a array gasket chamber for scan procedure.
Slides were scanned using the Agilent DNA Microarray Scanner using one color scan setting for 8x60k G3 array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green).
Gene expression control sample of 2h25min in 4 mg whole birch pollen exposure
Scanned images were analyzed with Feature Extraction Software Version 188.8.131.52 (Agilent) using default parameters (protocol GE1_1100_Jul11 and Grid: 072363_D_F_20200924) to obtain background and Processed Signal intensities.
Oct 05, 2021
Last update date
Mar 05, 2022
Technical University Munich & Helmholtz Center Munich