|
| Status |
Public on Aug 24, 2016 |
| Title |
subcapsular sinus, biologic replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
lymphatic endothelial cells, LCM
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6
tissue: subcapsular sinus
cell type: lymphatic endothelial cells, LCM
|
| Extracted molecule |
total RNA |
| Extraction protocol |
LCM: LNs were collected in SUPERase-In, placed onto a layer of OCT, Alexa fluor 488 anti-mouse CD31 and PE-Texas red anti-mouse F4/80 antibodies were applied to the frozen sections of lymph nodes. After washing the sections were dehydrated. LCMP was conducted on a PALM micro beam instrument to isolate CD31 labeled LEC cells. Micro-dissected cells were collected in adhesive caps. Altogether 50 cells were collected/experiment into Superamp lysis buffer. FACS sorting: LNs were digested in PBS containing liberase TM research grade and DNase I. For cell sorting the following antibodies were used: lineage (CD3e, CD11b, CD45R/B220, Erythroid Cells, Ly-6G and Ly-6C) cocktail,anti-CD73, and anti –podoplanin 7-AAD was used to exclude dead cells.1200 cells were sorted into fetal calf serum coated FACS tubes containing RPMI-1640 and 20% FCS. The samples were centrifuged and resuspended in SuperAmp Lysis Buffer.
|
| Label |
Cy3
|
| Label protocol |
Cy3-labeling was performed according to Miltenyi Biotec's undisclosed protocol
|
| |
|
| Hybridization protocol |
Cy3-labeled cDNAs were hybridized overnight (17h, 65C) to an Agilent Whole Mouse Genome Oligo Microarrays 8x60K using Agilent's recommended hybridization chamber and oven. The microarrys were washed once with the Agilent Gene Expression Wash Buffer 1 for 1 min at rt followed by a second wash with preheated Agilent Gene Expression Wash buffer 2 (37C) for 1 min.
|
| Scan protocol |
Signals were detected using Agilent's Microarray Scanner System. The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files.
|
| Data processing |
The signal intensities from the raw data lists were background corrected and normalized by dividing the intensity values by their median. The normalized intensities were log2-transformed.
|
| |
|
| Submission date |
Apr 28, 2015 |
| Last update date |
Aug 24, 2016 |
| Contact name |
Kati Elima |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Turku
|
| Department |
MediCity
|
| Street address |
Tykistökatu 6
|
| City |
Turku |
| ZIP/Postal code |
20520 |
| Country |
Finland |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE68371 |
Gene expression analysis of mouse lymphatic endothelial cells (LECs) from the subcapsular sinus (SS, afferent) and lymphatic sinus (LS, efferent) |
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