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| Status |
Public on May 08, 2014 |
| Title |
4: IL-10-non-secreting Th1 cells without activation of Notch signaling pathway derived from in vitro polarized TH1 cells |
| Sample type |
RNA |
| |
|
| Source name |
IL-10-negative Th1 cells without activation of the Notch signaling pathway
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6
genotype/variation: wildtype
cell type: primary naïve murine CD4+ cells
separation: erythrocyte lysis (EL)
magnetic cell sorting: depletion of CD25- cells and positive enrichment of CD4+ and CD62L+ cells
cell cultivation: Th1 polarizing conditions for 5 days
notch signaling pathway activation: not co-cultured with MACSi Beads covalently coated with recombinant mouse Notch ligand Delta-like 4 (Dll4)
cell restimulation: restimulated with PMA/Ionomycin
il10 staining: secreted IL-10 was stained using an IL-10 secretion assay (Miltenyi Biotec)
flow cytometric cell sorting: living CD4+ cells not secreting IL-10 (IL-10neg)
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| Biomaterial provider |
C57BL/6 mice were purchased from Jackson Laboratory.
|
| Treatment protocol |
Naïve T helper cells were enriched magnetically from lymph nodes and spleens of C57BL/6 wildtype mice after lysis of erythrocytes using EL buffer with the magnetic beads from the Multisort Kit from Miltenyi. Next, the enriched cells were cultured in an APC-free system under Th1 polarizing conditions in the absence or presence of a Notch inducing signal (Dll4). After 5 days of culture, cells were restimulated (PMA/Iono), and subjected to an IL-10 secretion assay. Dead cells were stained with propidiumiodide and CD4 using a fluorescently labeled antibody. These cells were then sorted by Aria or DIVA FACSorter machines. Chip 1 and 2 (with activation of the Notch signaling pathway): (1) Living CD4+ TH1 Notch IL-10 pos cells, (2) Living CD4+ TH1 Notch IL-10 neg cells. Chip 3 and 4 (without activation of the Notch signaling pathway): (3) Living CD4+ TH1 Control IL-10 pos cells, (4) Living CD4+ TH1 Control IL-10 neg cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instruction, including the recommended DNase digest. The purity and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip software and the amount was checked with a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).
|
| Label |
Biotin
|
| Label protocol |
Labeling of total RNA was performed using the two cycle GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturer's instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, two times amplified, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays.
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| |
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| Hybridization protocol |
The Mouse Genome 430 2.0 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
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| Scan protocol |
Arrays were scanned with an Affymetrix GeneChip Scanner 3000, using the GCOS software version 1.1.1. from Affymetrix.
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| Description |
Chip4_C57Bl6_CD4CD62L_TH1control_IL10neg_430_2
Primary naïve T helper cells were isolated from lymph nodes and spleens of C57BL/6 wildtype mice. Cells were enriched using the Multisort Kit from Miltenyi Biotec for CD25-CD4+CD62L+, and afterwards cultured under Th1 polarizing conditions (10 ng/ml rmIL-12, 10 µg/ml anti-IL-4 (11B11). For activation, 0.25E06 naïve T cells were co-cultured with 0.75E06 MACSi Beads that were coated with anti-CD3 and anti-CD28 (30 µg of total primary IgG antibody per 1.0E08 beads) prior to seeding. After 5 days in culture, the cells were restimulated with PMA/Ionomycin and subjected to an IL-10-secretion assay (Miltenyi Biotec) to obtain IL-10-non-secreting cells.
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| Data processing |
Data were analyzed using an Excel spreadsheet with original GCOS CHP file Signals. Transcripts up-regulated > 2 fold in IL-10 producing vs. non-producing Th1 cells and a detection p-value < 0.05 in at least one of the chips were filtered and analysed in silico. The AmiGO web interface (Ashburner et al., 2000) was used to filter those transcripts that were associated with a Gene Ontology term including "transcription" and/or their involvement in the biological process of "positive regulation of gene expression" (GO:0010628). Further validation of all these filtered transcripts was done via qPCR.
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| |
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| Submission date |
May 07, 2014 |
| Last update date |
May 04, 2016 |
| Contact name |
Pawel Durek |
| E-mail(s) |
pawel.durek@drfz.de
|
| Organization name |
Deutsches Rheuma-Forschungszentrum
|
| Street address |
Charitéplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE57417 |
Role of Blimp-1 in programing Th effector cells into IL-10 producers |
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