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| Status |
Public on Mar 26, 2014 |
| Title |
Th9 cells unstimulated rep2 |
| Sample type |
RNA |
| |
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| Source name |
naïve CD4 T cell cultured under Th9 conditions for 5 days
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: Th9
activation status: unstimulated
|
| Treatment protocol |
As described in growth protocol. For stimulated Th9 cultures, after five day sof culture, cells were stimulated with 1 ug/ml anti-CD3 for six hours.
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| Growth protocol |
Naive CD4+CD62L+ T cells were purified from spleens and lymph nodes by magnetic selection (Miltenyi Biotec). Naive CD4+ T cells (1 x 106 cells/ml complete RPMI 1640 medium) were activated with plate-bound anti-CD3 (2 mg/ml; 145-2C11; BioXcell) and soluble anti-CD28 (1 mg/ml; 37.51; BD Biosciences) and cultured under Th9 conditions (IL-4 [20 ng/ ml; PeproTech], TGF-b [2 ng/ml; R&D Systems], and anti–IFN-g [10 mg/ ml; XMG; BioXcell]); Th2 conditions (IL-4 and anti–IFN-g); Th1 con- ditions (IL-12 [5 ng/ml; R&D Systems], IL-2 [50 U/ml; PeproTech], and anti–IL-4 [10 mg/ml; 11B11; BioXcell]); and Treg conditions (TGF-b and anti–IL-4). After 3 d, cultures were expanded with fresh complete RPMI 1640 medium with IL-4 and TGF-b added to the Th9 cells; half the dose of IL-1b, IL-23, and IL-6 added to the Th17 cells; and IL-2 added to Tregs. RNA was harvested after 5 days of culture
|
| Extracted molecule |
total RNA |
| Extraction protocol |
performed by Miltenyi Biotec using standard RNA extraction protocols (NucleoSpin RNA II, Macherey-Nagel). Quality was assessed using electropherograms of agarose gels.
|
| Label |
Cy3
|
| Label protocol |
For the linear T7 based amplification step, 0.5 ug of total RNA was used to produce Cy3-labelled cRNA with amplification and labeling performed using the Agilent Quick Amp Labelling Kit. Incroporation rates were 12-17 fmol/ng.
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| |
|
| Hybridization protocol |
The hybridization procedure ws performed accoridng to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit. Cy3-labeled and fragmented cRNA was hybridzed overnight (17 hours 65°C) to the Agilent Whole Mouse Genome Oligo Microarrays 4x44K as recommended in the protocol. Microarrays were washed with Agilent Gene Expression Wash Buffer 1 for 1 min at room temp followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 at 37°C for 1 min. A final wash was performed with acetonitrile.
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| Scan protocol |
Fluorescence signals were detected using Agilent's Microarray Scanner System.
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| Description |
Gene expression in T cells cultured for 5d in Th9 conditions
Th9_non-stim_2
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| Data processing |
The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. For determination of differential gene expressionFES derived output data files were further analyzed using the Rosetta Resolver gene expression data analysis system.
The .txt Supplementary files were generated with Resolver Software.
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| |
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| Submission date |
Mar 07, 2013 |
| Last update date |
Mar 26, 2014 |
| Contact name |
Mark H Kaplan |
| E-mail(s) |
mkaplan2@iupui.edu
|
| Phone |
317-278-3696
|
| Organization name |
Indiana University
|
| Department |
Microbiology and Immunology
|
| Street address |
635 Barnhill Dr. MS 420
|
| City |
Indianapolis |
| State/province |
IN |
| ZIP/Postal code |
46202 |
| Country |
USA |
| |
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| Platform ID |
GPL7202 |
| Series (1) |
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