|
| Status |
Public on Dec 21, 2013 |
| Title |
neural-precursors_PSA-NCAM-enriched_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
[test] mESC derived neural precursors, PSA-NCAM enriched
|
| Organism |
Mus musculus |
| Characteristics |
enrichment: PSA-NCAM
cultivation: step 4
strain: C57BL/6
|
| Treatment protocol |
Cell suspensions containing approximately 3x106 cells were first labeled with anti-PSA-NCAM antibody conjugated to APC fluorochrome (Miltenyi Biotec). Then magnetic MicroBeads coupled to anti-APC antibody were applied. Cells were re-suspended in mESC medium and the cell suspension was loaded onto an MS-column (Miltenyi Biotec), which was placed in the magnetic field of a MACS Separator. The column was rinsed three times with 0.5 ml of medium. Magnetically isolated PSA-NCAM positive cells were retained in the column and eluted as the positively selected cell fraction after removing the column from the magnet. Immunomagnetic removal of dead cells prior enrichment with anti-PSA-NCAM was performed using the Dead Cell Removal Kit (Miltenyi Biotec). Isolated in vitro derived PSA-NCAM positive cells were seeded on polyornithine and laminin coated 24-well plates and further differentiated into neuronal lineage as describe above for in vitro analysis or were collected in medium at a concentration of 50000/ul and used for transplantation.
|
| Growth protocol |
Actin-eGFP C57BL/6 mESCs were propagated on Mitomycin-C (Sigma-Aldrich-Aldrich)-treated mouse fibroblasts in DMEM (Miltenyi Biotec) supplemented with 1mM nonessential amino acids (PAA), 1x NaPyruvate (PAA), 2mM L-Glutamin (PAA) 0.1 mM β-Mercaptoethanol, 1% Penicillin Streptomycin (PAA), 10% FCS (PAA) and 0.01% LIF (Miltenyi Biotec) (step 1). Cells were split every two days and plated at a concentration of 25000/cm2. Before embryoid bodies (EBs) differentiation (step 2), cells were harvested with 0,05% Trypsin (Sigma), subjected to feeder depletion with Feeder Removal MicroBeads Kit (Miltenyi Biotec) and then plated on nonadherent bacterial dishes for four days in LIF free EB medium containing 5% of Knockout serum replacement (Invitrogen) 5% FCS (PAA). EBs were then plated onto adhesive tissue culture surface. After 24h in culture, selection of neural progenitors cells was initiated in serum-free 1x ITS liquid media supplements (Sigma-Aldrich) plus 5µg/ml Fn (Biopure) supplemented DMEM medium (ITSFn, step 3). After 6 days (6d) of selection, cells were harvested with AccuMax (PAA), plated on polyornithine (Sigma-Aldrich) and laminin (Sigma-Aldrich) coated 24 well plates and cultivated for 5d (step 4) in Macs Neuro Medium (Miltenyi) supplemented with 1x N2 supplement (PAA), 1mM nonessential amino acids, 2mM L-Glutamin, 1% Penicillin Streptomycin, 1 µg/ml laminin, 10 ng/ml bFGF (Miltenyi). For neuronal differentiation (step 5) bFGF was removed to induce differentiation, N2 supplement was substituted with 1x B27 (PAA) supplement and 200 µM Ascorbic Acid was added. Cells were cultivated in neuronal differentiation medium for 7d.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1x 106 cells were harvested, rapidly frozen in liquid nitrogen and stored at -70 °C. RNA was isolated using standard RNA extraction protocols (Trizol). RNA quality and integrity were determined using the Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was quantified by measuring A260nm on the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| Label |
Cy5
|
| Label protocol |
Sample labeling was performed as detailed in the “Two-Color Microarray-Based Gene Expression Analysis protocol” (Agilent). For the linear T7-based amplification step, 100 ng of each total RNA sample was used. To produce Cy3- and Cy5-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| Channel 2 |
| Source name |
[control] mESC
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
|
| Treatment protocol |
Cell suspensions containing approximately 3x106 cells were first labeled with anti-PSA-NCAM antibody conjugated to APC fluorochrome (Miltenyi Biotec). Then magnetic MicroBeads coupled to anti-APC antibody were applied. Cells were re-suspended in mESC medium and the cell suspension was loaded onto an MS-column (Miltenyi Biotec), which was placed in the magnetic field of a MACS Separator. The column was rinsed three times with 0.5 ml of medium. Magnetically isolated PSA-NCAM positive cells were retained in the column and eluted as the positively selected cell fraction after removing the column from the magnet. Immunomagnetic removal of dead cells prior enrichment with anti-PSA-NCAM was performed using the Dead Cell Removal Kit (Miltenyi Biotec). Isolated in vitro derived PSA-NCAM positive cells were seeded on polyornithine and laminin coated 24-well plates and further differentiated into neuronal lineage as describe above for in vitro analysis or were collected in medium at a concentration of 50000/ul and used for transplantation.
|
| Growth protocol |
Actin-eGFP C57BL/6 mESCs were propagated on Mitomycin-C (Sigma-Aldrich-Aldrich)-treated mouse fibroblasts in DMEM (Miltenyi Biotec) supplemented with 1mM nonessential amino acids (PAA), 1x NaPyruvate (PAA), 2mM L-Glutamin (PAA) 0.1 mM β-Mercaptoethanol, 1% Penicillin Streptomycin (PAA), 10% FCS (PAA) and 0.01% LIF (Miltenyi Biotec) (step 1). Cells were split every two days and plated at a concentration of 25000/cm2. Before embryoid bodies (EBs) differentiation (step 2), cells were harvested with 0,05% Trypsin (Sigma), subjected to feeder depletion with Feeder Removal MicroBeads Kit (Miltenyi Biotec) and then plated on nonadherent bacterial dishes for four days in LIF free EB medium containing 5% of Knockout serum replacement (Invitrogen) 5% FCS (PAA). EBs were then plated onto adhesive tissue culture surface. After 24h in culture, selection of neural progenitors cells was initiated in serum-free 1x ITS liquid media supplements (Sigma-Aldrich) plus 5µg/ml Fn (Biopure) supplemented DMEM medium (ITSFn, step 3). After 6 days (6d) of selection, cells were harvested with AccuMax (PAA), plated on polyornithine (Sigma-Aldrich) and laminin (Sigma-Aldrich) coated 24 well plates and cultivated for 5d (step 4) in Macs Neuro Medium (Miltenyi) supplemented with 1x N2 supplement (PAA), 1mM nonessential amino acids, 2mM L-Glutamin, 1% Penicillin Streptomycin, 1 µg/ml laminin, 10 ng/ml bFGF (Miltenyi). For neuronal differentiation (step 5) bFGF was removed to induce differentiation, N2 supplement was substituted with 1x B27 (PAA) supplement and 200 µM Ascorbic Acid was added. Cells were cultivated in neuronal differentiation medium for 7d.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1x 106 cells were harvested, rapidly frozen in liquid nitrogen and stored at -70 °C. RNA was isolated using standard RNA extraction protocols (Trizol). RNA quality and integrity were determined using the Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was quantified by measuring A260nm on the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| Label |
Cy3
|
| Label protocol |
Sample labeling was performed as detailed in the “Two-Color Microarray-Based Gene Expression Analysis protocol” (Agilent). For the linear T7-based amplification step, 100 ng of each total RNA sample was used. To produce Cy3- and Cy5-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| |
|
| |
| Hybridization protocol |
Hybridisation was performed as detailed in the “Two-Color Microarray-Based Gene Expression Analysis protocol” (Agilent). Briefly, 825 ng of the corresponding Cy3- and Cy5-labeled fragmented cRNA were combined and hybridized overnight (17 hours, 65 °C) to Agilent Whole mouse Genome Oligo Microarrays 4x44K Version 2 using Agilent’s recommended hybridization chamber and oven. Finally, the microarray was washed once with the Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 (37 °C) for 1 min. The last washing step was performed with acetonitrile.
|
| Scan protocol |
The Agilent Feature Extraction Software (FES) version 10.7.3.1 was used to read out and process the microarray image files.
|
| Description |
Sample name: step 4 enriched 2
Agilent Feature Extraction file:
252665512019_S01_GE2_105_Jan09_1_2.txt
|
| Data processing |
FES derived output data files were further processed using the Rosetta Resolver® gene expression data analysis system (Rosetta Biosoftware).
|
| |
|
| Submission date |
Jan 16, 2012 |
| Last update date |
Dec 22, 2013 |
| Contact name |
Stefan Tomiuk |
| E-mail(s) |
[email protected]
|
| Organization name |
Miltenyi Biotec GmbH
|
| Department |
Bioinformatics
|
| Street address |
Friedrich-Ebert-Str. 68
|
| City |
Bergisch-Gladbach |
| ZIP/Postal code |
51429 |
| Country |
Germany |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE35125 |
Immunomagnetic purification of mES derived neuronal precursors increases neuronal migration and differentiation in vivo |
|
