|
| Status |
Public on Jun 25, 2024 |
| Title |
S_6_B1_2 |
| Sample type |
RNA |
| |
|
| Source name |
A549
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A549
treatment: Dimethylamine
concentration: 14.9 ppm
replicate: 2
exposure plate: 1
group: LD
|
| Treatment protocol |
To mimic the exposure situation of the epithelium in the in vivo lung the most common approach used is the air-liquid-interface (ALI) technique based on cell cultures on microporous membranes. Therefore, cells were initially cultivated under their cell type specific conditions in 75cm² culture flasks using submerged conditions. Culture medium was changed every to 2-3 days. Before reaching 80% confluence, cells were subcultivated. During a cell passage an aliquot of the cells was then seeded on microporous membranes (Inserts, BD Falcon; 0.4µm pore size; growth area ~1cm²). Cells were further cultivated on the membranes for approximately 72hrs until they reached a confluent monolayer as inspected by light microscopy. Serum was removed from the culture during a medium change 18hrs before exposure. Previous to the exposure with the model substances, residual liquid from the apical side of each cell monolayer was gently removed. During the treatment, cells were nutrified by culture media from beneath the membrane solely while being exposed to the test substances from the top.
|
| Growth protocol |
The A549 cell line was purchased from a commercial supplier (ATCC; LGC Promochem). Cells were routinely taken from a stock pool and grown in 75cm2 flasks by use of Dulbecco´s MEM medium (Seromed, Berlin) supplemented with 10% FCS and antibiotics. Cells were passaged every 3 to 4 days. During each passage beside continuous microscopic observation, cell quality and quantity were checked by use of an electronic cell counter (CASY® Cell Counter + Analyser System; Schärfe System, Reutlingen, Germany).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated and purified using the RNeasy MiniKit (Qiagen) and treated with DNase (Qiagen). RNA concentration (A260) and purity (A260/A280 ratio) were measured by spectrophotometry (NanoDrop™ 2000 Spectrophotometer, software version 1.6.198, ThermoFisher Scientific). RNA integrity number (RIN) was evaluated using an Agilent 2100 Bioanalyzer® (Agilent Technologies). All RNA samples showed very good quality as indicated by high RIN values between 9.0 and 10.0.
|
| Label |
biotin
|
| Label protocol |
Affymetrix GeneChip™ Whole Transcript (WT) PLUS Reagent Kit according to the manufacturer’s recommendation (ThermoFisher).
|
| |
|
| Hybridization protocol |
GeneChip™ Human Clariom™ D Arrays according to the manufacturer’s recommendation (ThermoFisher).
|
| Scan protocol |
Affymetrix GeneChip™ Command Console Software with .cel files as data output.
|
| Data processing |
Array data were analyzed with the Transcriptome Analysis Console (TAC) Software 4.0 (ThermoFisher).
|
| |
|
| Submission date |
Aug 15, 2023 |
| Last update date |
Jun 25, 2024 |
| Contact name |
Matthias M Wehr |
| Organization name |
Fraunhofer ITEM
|
| Street address |
Nikolai-Fuchs-Str. 1
|
| City |
Hannover |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL23126 |
| Series (2) |
| GSE240871 |
M3.3 Clariom D Arrays Dimethylamin |
| GSE240878 |
A549 in vitro cell culture exposed to 3 concentrations of Dimethylamin in an Air-liquid Interface. |
|
