|
| Status |
Public on Oct 06, 2010 |
| Title |
Donor 2, mock-infected human type I-like alveolar epithelial cells at 8h |
| Sample type |
RNA |
| |
|
| Source name |
Differentiated human type I-like alveolar epithelial cells mock infected at 8h post-infection time
|
| Organism |
Homo sapiens |
| Characteristics |
individual: donor 2
tissue: lung
cell type: differentiated type I-like alveolar epithelial cells
infection: mock
time point: 8h post-infection
|
| Treatment protocol |
Differentiated type I-like alveolar epithelial cells were mock infected, or infected with pdmH1N1 or seasonal H1N1 at a multiplicity of infection (MOI) of two. Total RNA was extracted from cells after 8h post-infection.
|
| Growth protocol |
Isolation of primary human alveolar type II alveolar epithelial cells: Primary type II alveolar epithelial cells were isolated using human non-malignant lung tissue obtained from patients undergoing lung resection. After removing visible bronchi, the lung tissue was minced into pieces of > 0.5 mm thickness using a tissue chopper and washed with balanced salt solution containing Hanks' balanced salt solution with 0.7 mM sodium bicarbonate at pH 7.4 for 3 times to partially remove macrophages and blood cells. The tissue was digested using a combination of 0.5% trypsin and 4 U/ml elastase for 40 min at 37°C in a shaking water-bath. The digestion was stopped by adding DMEM/F12 medium with 40% FBS in and DNase I (350 U/ml). Cell clumps were dispersed by repeatedly pipetting the cell suspension for 10 min. A disposable cell strainer (gauze size of 50 μm) was used to separate large undigested tissue fragments. The single cell suspension in the flow-through was centrifuged and resuspended in a 1:1 mixture of DMEM/F12 medium and small airway basal medium (SABM)supplemented with 0.5 ng/ml epidermal growth factor (hEGF), 500 ng/ml epinephrine, 10 μg/ml transferrin, 5 μg/ml insulin, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyronine, 0.5 μg/ml hydrocortisone, 30 μg/ml bovine pituitary extract and 0.5 mg/ml BSA together with 5% FBS and 350 U/ml DNase I. The cell suspension was plated on plastic flask and incubated in a 37°C water-jacketed incubator with 5% CO2 supply for 90 min. The non-adherent cells were layered on a discontinuous cold Percoll density gradient (densities 1.089 and 1.040 g/ml) and centrifuged at 25×g for 20 min without brake. The cell layer at the interface of the two gradients was collected and washed four times with BSS to remove the Percoll. The cell suspension was incubated with magnetic beads coated with anti-CD14 antibodies at room temperature for 20 min under constant mixing. After the removal of the beads using a magnet (MACS CD14 MicroBeads), cell viability was assessed by trypan-blue exclusion. The purified alveolar epithelial cell suspension was resuspended in small airway growth medium supplemented with 1% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were maintained in a humidified atmosphere (5% CO2, 37°C) under liquid-covered conditions, and growth medium was changed daily starting from 60 h after plating the cells.
Type I-like alveolar epithelial cell differentiation: The purified type II alveolar epithelial cell pellet (passage 1 or 2) was resuspended in medium and cultured for 14 to 20 days with the small airway culture medium SAGM. The cells spread to form a confluent monolayer and the culture medium was changed every 48 h before used for virus infection experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the RNEasy Mini kit (Qiagen) according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Total RNA was labeled employing the Whole Transcript (WT) Sense Target Labeling Assay according to the manufacturer’s instructions (Affymetrix).
|
| |
|
| Hybridization protocol |
The samples were hybridized to Human Gene 1.0 ST microarrays according to standardized protocols from Affymetrix.
|
| Scan protocol |
The samples were scanned on a GeneChip Scanner 3000 7G (Affymetrix).
|
| Description |
D2_mock
Gene expression data of mock-infected type I-like alveolar epithelial cells at 8 h post-infection time.
|
| Data processing |
The microarray data were preprocessed with Affymetrix Expression Console software using the Affymetrix default Robust Multichip Analysis (RMA) sketch algorithm workflow.
|
| |
|
| Submission date |
Oct 05, 2010 |
| Last update date |
Oct 05, 2010 |
| Contact name |
SMY Lee |
| Organization name |
The University of Hong Kong
|
| Street address |
21 Sassoon Road
|
| City |
Hong Kong |
| ZIP/Postal code |
HKG |
| Country |
Hong Kong |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE24533 |
Expression data of influenza A-infected human type I-like alveolar epithelial cells |
|
