|
| Status |
Public on Dec 01, 2010 |
| Title |
B cells from mice treated with MPL - rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
MPL-treated_2
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: B-cell
stimulation: MPL (TLR4 ligand)
time: 7 days post-treatment
cell markers: TCRbeta-CD11b-CD19+IgD-IgG+ B cells
|
| Treatment protocol |
8-12 week old C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were immunized with 10µg of ovalbumin antigen in nanoparticles (suspended in 200µl of PBS), subcutaneously at the base of the tail. PLGA nanoparticles containing 37.5µg of MPL were used with the protein encapsulated particles. At day 7 post-immunization, activated, isotype switched B cells (TCRβ-CD11b-CD19+IgD-IgG+ cells) were isolated from draining lymph nodes (pooled) from immunized mice (n=5), sorted by FACS and RNA was isolated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from sorted B-cells (1.8 x 10^4 to 1.2 x 10^6 cells) was purified using Trizol® (Invitrogen, Life Technologies Corporation).
|
| Label |
Biotin
|
| Label protocol |
All RNA samples were checked for purity using a ND-1000 spectrophotometer (NanoDrop Technologies) and for integrity by electrophoresis on a 2100 BioAnalyzer (Agilent Technologies). The samples were amplified using the Nugen WT Pico Kit (NuGEN Technology) and the target reactions were run with 50ng of Total RNA. The amplification products were processed through the EXON Module (NuGEN Technology) which creates sense-strand cDNA targets. The sense strand cDNA Targets were then fragmented and labeled using NuGEN’s FL-Ovation™ cDNA Biotin Module V2 (NuGEN Technology).
|
| |
|
| Hybridization protocol |
Labeled targets were hybridized to GeneChip® Mouse Gene 1.0ST arrays (Affymetrix, Inc.), following Standard Nugen Protocols for target hybridization to the Affymetrix Gene Arrays. The hybridizations were run for 16 hours, 45oC, 60RPM in an Affymetrix Hybridization Oven 640. The Cartridge arrays were washed and stained using the Affyemtrix Fluidics Stations 450, following Affymetrix protocols.
|
| Scan protocol |
Scanning was performed on an Affymetrix GeneChip 3000 7G scanner, and Affymetrix GCOS software was used to perform image analysis and generate raw intensity data.
|
| Description |
Gene expression from FACS-sorted B cells
|
| Data processing |
Probe sets of all 6 samples were normalized by RMA, which includes global background adjustment and quantile normalization. program-name = Expression Console; affymetrix-algorithm-name = rma-gene-default.
Each set of samples was subsequently normalized by z-score (number of standard deviation from mean) and treated as biological replicates.
|
| |
|
| Submission date |
Nov 29, 2010 |
| Last update date |
Dec 01, 2010 |
| Contact name |
Helder I. Nakaya |
| E-mail(s) |
[email protected]
|
| Phone |
+551126481130
|
| Organization name |
Universidade de São Paulo
|
| Lab |
csbiology.com
|
| Street address |
AVENIDA PROFESSOR LINEU PRESTES, 580, Block 17
|
| City |
SÃO PAULO |
| State/province |
SÃO PAULO |
| ZIP/Postal code |
05508000 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE25677 |
Sorted B cells from mice treated with MPL and/or R837 |
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