|
| Status |
Public on Sep 21, 2017 |
| Title |
E12-HpInfected-Rep2 |
| Sample type |
RNA |
| |
|
| Source name |
HT29-MTX-E12 cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HT29-MTX-E12
cell line_1: Derivative of the human cell line HT29 from an original adenocarcinoma of the colon
cell line_2: Selected from HT29-MTX on the basis of tight junction formation and adherent mucus production, having many of the characteristics of a gastric cell (Behrens et al., 2001, DOI: 10.1023/A:1010974909998)
infection: H. pylori strain 26695
|
| Treatment protocol |
On day 20 E12 cells in transwells were washed with HBSS and 2 ml antibiotic-free complete DMEM media was added to the base of each well and 1 ml of RPMI to the apical surface of each well. On day 21 200ml bacteria (6 x 108) were added to each well at an MOI of 300. Plates were incubated at 37 oC for 24 h. Replicate transwells were used to determine the number of bacteria (CFU/ml) that were associated with the cells. Cells were harvested for RNA isolation.
|
| Growth protocol |
E12 cells were maintained in flasks in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % foetal calf serum, 2 mM L-glutamine, 1 % nonessential amino acids and antibiotics (penicillin (50 U/ml) and streptomycin (50 μg/ml). Cells (2 x 106) were seeded on transwell filters (0.4 micometer pore polycarbonate membrane insert) in 6-well plates. Cells were incubated at 37 oC in a humidified 5 % CO2 atmosphere and media was added every second day. TEER measurements were taken at 20 days. Antibiotics were removed from the cells at least 24 h before infection assays.
Bacteria were plated on Columbia blood agar (Oxoid) plates and grown for 48 h in a microaerophilic environment using gas packs (Oxoid, BR0038). Bacteria were harvested from plates into Brucella broth and the OD600 adjusted to 0.1 and grown in T25 tissue culture flasks at 37 oC under microaerophilic conditions with shaking at 70-80 rpm until the OD600 reached 0.6. Gram staining of bacterial cultures was used to check coccoid levels were less than 5 %. Washed bacteria were resuspended to an OD600 of 1.8 with antibiotic-free RPMI.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA for microarray hybridizations was isolated by TRIzol extraction followed by standard RNA purification protocols using the RNeasy Mini Kit (Qiagen). RNA quality control was carried by a commercial company (DNA Vision, Charleroi, BE) using an Agilent Bioanalyzer 2100. The amount of eukaryotic RNA was corrected across samples using the 28S RNA peak because of the variable presence of prokaryotic RNA (infecting Helicobacter pylori).
|
| Label |
Biotin
|
| Label protocol |
Affymetrix 3' IVT Express Protocol. Carried out by DNA Vision, Charleroi, BE starting from 100 ng of eukaryotic RNA
|
| |
|
| Hybridization protocol |
All hybridizations were carried out using the hybridization and wash solutions as recommended in the Affymetrix 3' IVT Express Protocol. Carried out by DNA Vision, Charleroi, BE.
|
| Scan protocol |
Agilent Technologies Scanner G2505C (5 micron reolution). Carried out by DNA Vision, Charleroi, BE
|
| Description |
E12_HP_5
|
| Data processing |
Carried out by DNA Vision, Charleroi, BE. Images were processed using GenePix Pro 6.1.0.4. Spot intensities were normalized and analyzed in GeneSpring (Ver 12.3). Data were log transformed and quantile normalized. Subsequently all normalized data was baseline transformed to the median of all samples.
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| |
|
| Submission date |
Oct 29, 2015 |
| Last update date |
Dec 01, 2017 |
| Contact name |
Michael Cairns |
| E-mail(s) |
michael.cairns@nuigalway.ie
|
| Phone |
0035391492094
|
| Organization name |
NUI Galway
|
| Department |
NCBES
|
| Lab |
Glycoscience Group
|
| Street address |
University Road
|
| City |
Galway |
| ZIP/Postal code |
NA |
| Country |
Ireland |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE74492 |
Glycosylation-related gene expression in the mucus-secreting gastrointestinal cell line HT29-MTX-E12 in response to infection by Helicobacter pylori |
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