|
| Status |
Public on Jun 30, 2007 |
| Title |
donor2_CD8_stage3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
human blood (naïve)
|
| Organism |
Homo sapiens |
| Characteristics |
Pool of three independently FACS sorted naive CD8+ T lymphocytes (CD45RA+/CCR7+/CD28+/CD27+)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
SuperAmp Service, Miltenyi
|
| Label |
Cy13
|
| Label protocol |
For the generation of amplified cDNA, 1- 10000 cells were collected in 6.4 µl lysis buffer (including detergent, tRNA and Protease), the mRNA extracted using magnetic beads and transcribed into cDNA using tagged random and oligo(dT) primer. First strand c
|
| |
|
| Channel 2 |
| Source name |
human blood (highly differentiated)
|
| Organism |
Homo sapiens |
| Characteristics |
FACS sorted_CD8_stage3_(CD27+/CD28+/CCR7-)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
SuperAmp Service, Miltenyi
|
| Label |
Cy5
|
| Label protocol |
For the generation of amplified cDNA, 1- 10000 cells were collected in 6.4 µl lysis buffer (including detergent, tRNA and Protease), the mRNA extracted using magnetic beads and transcribed into cDNA using tagged random and oligo(dT) primer. First strand c
|
| |
|
| |
| Hybridization protocol |
PIQOR Microarray Immunology, human, sense (Miltenyi) hybridization was performed according to the manufactures instructions using an automized hybridization machine (a-Hyb, Miltenyi Biotec). Briefly, volume of labeled sample was adjusted to 100 µl with nu
|
| Scan protocol |
Image capture and signal quantification of hybridized PIQOR(TM) microarrays were done with the ScanArrayLite4000 Microarray Scanner (PackardBiosciences) and ImaGene software Version 5.0 (BioDiscovery, Los Angeles, CA, USA).
|
| Description |
For cell surface, a panel of titrated anti-human antibodies was added to freshly isolated PBMC for 15 min at room temperature (RT). For intracellular staining, cells were washed, permeabilised with FACSTM Permeabilisation buffer (Becton Dickinson), and incubated for 15 min at RT in the dark with antibodies for intracellular molecules (Perforin, Granzymes A and B). Cells were then washed and stored at 4°C until flow cytometry analysis or sorting was performed. Samples were analyzed on a Becton Dickinson LSRII. For cell sorting, peripheral blood mononuclear cells (PBMC) were obtained by Ficoll s
|
| Data processing |
Signal quantification of the scanned PIQOR(TM) microarrays was done using ImaGene software Version 5.0 (BioDiscovery, Los Angeles, CA, USA). Local background was subtracted from the signal to obtain the net signal intensity and the Cy5/Cy3-ratio. The latter was only calculated from: (a) unflagged spots (b) showing a fluorescent intensity in one of the two channels higher than an additional lower cutoff. This lower cutoff is defined as twice the global background. Subsequently, the mean Cy5/Cy3-ratio of validated spots representing the same cDNA was computed. The mean ratios were normalised using the Median method.
|
| |
|
| Submission date |
Jan 12, 2007 |
| Last update date |
Mar 21, 2007 |
| Contact name |
Frank Huebel |
| E-mail(s) |
[email protected]
|
| Fax |
+49 221 950 48 48
|
| URL |
http://www.miltenyibiotec.com
|
| Organization name |
Miltenyi Biotec GmbH, MACS Molecular Business Unit
|
| Department |
MACS Molecular 9
|
| Street address |
Stoeckheimer Weg 1
|
| City |
COLOGNE |
| State/province |
NRW |
| ZIP/Postal code |
50829 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4725 |
| Series (1) |
| GSE6732 |
Sensitive expression profiling of T-cell subsets reveals parallel post-thymic differentiation for CD4+ and CD8+ lineages |
|
