|
| Status |
Public on Apr 11, 2016 |
| Title |
WT_IAV_AECII_d3_repl_2 |
| Sample type |
RNA |
| |
|
| Source name |
AECII from d3 IAV infected WT C57BL/6 mouse
|
| Organism |
Mus musculus |
| Characteristics |
genotype: WT
treatment: IAV infection
days after infection: 3
cell material: alveolar type II epithelial cells
|
| Treatment protocol |
Mice were used for experiments at an age between 8 and 12 weeks and control groups were age- and sex-matched. Mice were intranasally infected with a lethal dose of IAV PR8/A/34(H1N1) in a volume of 40 µl. For anesthesia, the animals were intraperitoneally injected with a mixture of ketamine/xylazine and the virus in PBS or PBS alone for control groups was administered onto the nostrils to be taken up upon breathing. Samples (lungs, AECII cells) were prepared at indicated time points.
|
| Growth protocol |
For AECII isolation lungs were perfused with PBS through the heart and instilled with 2 ml dispase solution and 500 µl liquefied low-melt agarose (1 %) through the trachea using a 22 G indwelling cannula. When the agarose was solidified, lungs were excised and incubated in 2 ml dispase solution for 45 min at room temperature. The lung tissue of single lungs was then disintegrated using forceps and incubated at room temperature for 10 minutes gently rocking in DMEM medium supplemented with DNase and anti CD16/32 antibody for blocking of Fc receptors while. Lung cell suspensions were filtered subsequently through nylon meshes of 100 µm, 70 µm, 45 µm and 30 µm size. At this step, 5 mice per group were pooled for each experimental condition. Following filtration, cells were stained for CD16/32, F4/80, CD11b, CD11c, CD19 and CD45. Primary murine AECII were isolated by negative selection by sorting for SSChigh cells negative for the antibody-staining using a FACS sorter and stored in RNAlater buffer (Qiagen). In case of whole lung material lungs were perfused with PBS through the heart and stored in RNAlater buffer.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. DNA was digested by using the RNase-Free DNase Set (Qiagen). RNA was eluted in 100 μl nuclease-free water and further concentrated by ethanol precipitation. RNA integrity was tested by using the Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany) with an Agilent RNA 6000 Nano/Pico Kit (Agilent Technologies).
|
| Label |
biotin
|
| Label protocol |
Labeling was performed using the GeneChip HT 3' IVT Express Kit (Affymetrix) according to manufacturers instructions.
|
| |
|
| Hybridization protocol |
Hybridization and washing was performed according to Affymetrix company's recommendations. GeneChips were washed in an Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GCS 3000 scanner and GCOS v1.1.1 software.
|
| Data processing |
Microarray data were analyzed using GeneSpring GX software (Agilent technologies). Data were summarized and normalized with the RMA algorithm.
|
| |
|
| Submission date |
Apr 23, 2014 |
| Last update date |
Apr 11, 2016 |
| Contact name |
Andreas Jeron |
| E-mail(s) |
andreas.jeron@helmholtz-hzi.de
|
| Organization name |
Helmholtz Center for Infection Research
|
| Department |
Viral Immunology
|
| Lab |
Immune Regulation
|
| Street address |
Inhoffenstraße 7
|
| City |
Braunschweig |
| State/province |
Nierdersachsen |
| ZIP/Postal code |
38124 |
| Country |
Germany |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE57008 |
Transcriptomics of murine ex vivo isolated alveolar type 2 epithelial cells from Influenza A respiratory infection |
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