|
| Status |
Public on Dec 21, 2011 |
| Title |
normal mouse dendritic sorted CD11cint F4/80int rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
normal mouse dendritic sorted CD11cint F4/80int
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
|
| Treatment protocol |
After removing extra-intestinal fat tissue and blood vessels, colons were flushed of their luminal content with HBSS, opened longitudinally, and cut into 2 cm pieces and incubated with HBSS containing 0.015% DTT (15 min, 37°C in a shaking water bath). Epithelial cells and mucus were removed by extensive washes in HBSS, 5% FCS, 25 mM Hepes, before colons were cut into smaller 2mm2 pieces and digested in complete Iscoves media containing Liberase TL (167 µg/mL) and DNAse I (30 µg/mL) (Roche # 05401020001 and #10104150001) for 60 min at 37°C in a shaking water bath. The digested cell suspension was then passed through 100- and 40-µm cell strainers and resuspended in 1.077 g/cm3 iso-osmotic NycoPrep medium (Accurate Chemical & Scientific Corp.). After centrifugation at 1,600 g for 15 min, the low-density fraction was collected. Cells were then stained with Fc block [CD16/32 (2.4G2)] and antibodies to CD11c(HL-3), MHC-II (AF6-120.1), F4/80 mAb (BM8), CD11b (M1/70), CD103 (M290), as well as antibodies to B cell and T cell lineages (referred here after as “lin”): CD19 (RA3-6B2), CD3 (145-2C11), TCRβ (H57-597), TCRγδ (eBioGL3) all from eBioscience. After pre-gating on live, MHC-IIhigh lin- cells, the indicated dendritic cell and macrophage subsets were sorted using a BD Biosciences FACSVantage or FACSAria machine.
|
| Growth protocol |
Rag2–/– mice were injected i.p. with 1.5 x 105 CD4+ CD45RBhigh + 1.5 x 105 CD4+ CD45RBlow T cells (non-colitic CD45RBhigh+low mice), isolated from the spleens of wild type C57BL/6 mice.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was isolated using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. RNA quantity /quality were assessed using the Agilent 2100 Bioanalyzer. RNA was reverse transcribed into cDNA (SuperScript III; Invitrogen)
|
| Label |
biotin
|
| Label protocol |
The cDNA was prepared and labeled according to standard manufacturer protocols (Affymetrix)
|
| |
|
| Hybridization protocol |
Samples were hybridized onto Affymetrix GeneChip Arrays (Affymetrix) according to manufacturer's recommendations
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 7GPlus.
|
| Description |
Gene expression data from normal mouse dendritic sorted CD11cint F4/80int
|
| Data processing |
The data were analyzed with GCOS 1.4 and EC 1.1 using Affymetrix default analysis and normalization method.
|
| |
|
| Submission date |
Mar 09, 2011 |
| Last update date |
Dec 21, 2011 |
| Contact name |
Dan Sturdevant |
| E-mail(s) |
dsturdevant@niaid.nih.gov
|
| Phone |
4063639248
|
| Organization name |
NIH
|
| Department |
NIAID
|
| Lab |
RTS
|
| Street address |
903 S 4th street
|
| City |
Hamilton |
| State/province |
MT |
| ZIP/Postal code |
59840 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE27859 |
Inflammation switches the differentiation program of Ly6Chi monocytes from anti-inflammatory macrophages to inflammatory dendritic cells in the colon |
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