|
| Status |
Public on Dec 25, 2016 |
| Title |
IL4 DC-ND311-Medium-6h-in vitro |
| Sample type |
RNA |
| |
|
| Source name |
IL4 DC-Medium-6h-in vitro
|
| Organism |
Homo sapiens |
| Characteristics |
^sample: IL4 DC_ND311_Medium_6h
cell population: IL4 DC
donor: ND311
stimulus: Medium
timepoint: 6h
experiment: IL4 DC vs TIV-09/MIV-09
sample type: in vitro
|
| Treatment protocol |
Cells were cultured in sterile 50 mL tissue culture bags (AFC, Gaithersburg, MD) at a density of 1x10^6 cells/mL along with GM-CSF at 10 ng/mL and IL-4 (R&D systems, Minneapolis, MN) at 50 ng/mL for 6 days. Cells were supplemented with additional cytokine doses on days 2 and 4. On day 6, DCs were harvested and further cultured in 200 µL of RPMI 1640 supplemented with (1% glutamine, 1% penicillin/streptomycin, 1% HEPES, 1% non-amino-acids, 1% sodium pyruvate and 0.1% β-mercaptoethanol) (Invirogen) and10% fetal bovine serum (FBS) at a density of 1x105 cells/mL in 96-well plates. TIV-09 (6 µL/mL) and/or MIV-09 (6 µL/mL) (Sanofi Pasteur, Swiftwater, PA) was added, and cells were incubated at 37°C for 0, 6, 12, 24 and 48h. Supernatants were stored at -80°C.
|
| Growth protocol |
Monocyte-derived DCs (moDCs) were generated from monocytes isolated from healthy donor apheresis fractionated by elutriation. Monocytes were enriched by negative selection using EasySep Human Monocyte Enrichment without CD16 Depletion Kit (Stemcell technologies, Vancouver, Canada) and cultured in CellGro DC media (CellGenix, Freiburg, Germany) with 1% penicillin/streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After vaccine stimulation, cells were lysed in RLT buffer (RNeasy Plus Mini Kit, Qiagen, Hercules, CA), 1% β-mercaptoethanol and stored at -80oC. Total RNA was isolated from cell lysates according to manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
RNA from samples passing quality control was amplified and labeled with Illumina TotalPrep RNA amplification kit (Illumina, San Diego, CA). RNA integrity was assessed using an Agilent 2100 Analyzer (Agilent Technologies, Palo Alto, CA).
|
| |
|
| Hybridization protocol |
Biotinylated complementary RNA (cRNA) was hybridized to Illumina Human-6 Beadchip Array version 2 using Illumina standard protocol
|
| Scan protocol |
Hybridized chips were scanned in Illumina Beadstation 500.
|
| Description |
5543531039_K
|
| Data processing |
Fluorescent hybridization signals were assessed with Beadstudio software (Illumina). Statistical analysis and hierarchical clustering were performed using GeneSpring 7.3.1 software (Agilent Technologies)
|
| |
|
| Submission date |
May 20, 2016 |
| Last update date |
Mar 16, 2023 |
| Contact name |
Nicole Baldwin |
| E-mail(s) |
Nicole.Baldwin@BSWHealth.org
|
| Organization name |
Baylor Research Institute
|
| Street address |
3434 Live Oak St
|
| City |
Dallas |
| ZIP/Postal code |
75204 |
| Country |
USA |
| |
|
| Platform ID |
GPL10558 |
| Series (2) |
| GSE81691 |
Influenza Vaccines Differentially Regulate the Interferon Response in Human Dendritic Cell Subsets [HumanHT-12 V4.0 Beadchip Experiments] |
| GSE81692 |
Influenza Vaccines Differentially Regulate the Interferon Response in Human Dendritic Cell Subsets |
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