|
| Status |
Public on May 01, 2011 |
| Title |
neurons_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
primary cortical neurons
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
developmental stage: E15
tissue: neuronal
experimental treatment: untreated
|
| Treatment protocol |
NMDA excitotoxic death was induced by exposure of neurons to 30 μM NMDA for 24h. N-methyl-d-aspartate (NMDA) (Sigma-Aldrich; M3262) was diluted in water at 340 μM. A final concentration of 30μM NMDA was added to cultured neurons to induce excitotoxic death; (~25% as measured by Hoechst staining and 35% as estimated by LDH assays). Neuron injury was assessed by measurement of LDH release assay (Koh & Choi 1987), WST-1 assay (Roche) and evaluation of nuclear morphology with Hoechst 33342 staining (Cheng et al 1994). Cortical neurons plated in 24 well dishes were fixed with 4% paraformaldehyde and acetone, stained with the Hoechst dye and visualized under a fluorescence microscope as previously described (Farinelli and Greene, 1996; Stefanis et al., 1998). Apoptotic nuclei were identified as nuclei with chromatin margination along the nuclear membrane or with chromatin condensation, with formation of discrete homogeneous chromatin clumps. The percentage of apoptotic nuclei was counted for each condition at 40× magnification in four separate fields of cells and is reported as the mean ± SEM. Measurement of lactate dehydrogenase (LDH) released from damaged neurons into the culture medium was performed as described previously (Koh & Choi 1987) and culture media of untreated neurons was used for normalization. The WST-1 assay was used according to the manufacturer’s protocol to measure the viability of neuronal cells in bFGF neutralization experiments. Once in the interior of metabolic active cells, WST-1 is reduced by mitochondrial succinate-tetrazolium reductase to produce the colorimetrically differentiated formazan product (K. Cesnulevicios et al., 2006). MSC pre-treatment of DIV5 neurons was performed by culture of neurons (400,000 cells/cm2) with conditioned medium from MSC (20.000 cells/cm2 in high-glucose DMEM) (MSC cm) for 24 h. High-glucose DMEM or conditioned medium from NIH/3T3 fibroblasts (12.5 x 104/ml) (3T3 cm) was used as control.
|
| Growth protocol |
MSC were isolated from the bone marrow of C57BL/6 or TgN(act-EGFP)OsbC14-Y01-FM131 (FM131) mice using plastic adherence capacity as previously described {Friedenstein 1995 68 /id}. Briefly, bone marrow cells were dissociated and cultured at 5x105/cm2 in minimum essential medium alpha (α-MEM, Gibco/BRL) supplemented with 10% FCS (Gibco/BRL), L-Glutamine 2mM (Sigma-Aldrich), antibiotics and 2ng/ml recombinant FGF2 with frequent medium changes until they reached confluency. Cells were harvested with 0.05% v/v trypsin/1mM EDTA (Gibco/BRL) and re-plated at 2x104/cm2. MSC were used for experiments after the 6th passage when the purity of MSC (Sca1+CD44+CD11b-) as assessed by FACS analysis was 98%. FACS analysis was performed using antibodies to the surface markers Sca1 (clone E13-161.7), CD44 (clone IM7) and CD11b (clone M1/70) (BD Biosciences) and a FACSCalibur with CellQuest software (BD Biosciences). MSC conditioned medium (MSC cm) was prepared by incubation of MSC for 24 h (2x104/cm2) in high-glucose DMEM. Dissociated neocortical cell cultures were prepared from E15 mice as previously described (Taoufik et al 2007). Briefly, cells were plated onto poly-D-lysine/laminin (Sigma-Aldrich)-coated dishes at 400,000 cells/cm2. Cells were maintained in DMEM supplemented with 4.5 g/L glucose (Sigma-Aldrich, # D5671), 5% foetal bovine serum (Invitrogen), 5% horse serum (Invitrogen) and 2mM glutamine (Invitrogen) (high-glucose DMEM). On day 3 in vitro (DIV3), 10 μM arabinose-C (Sigma-Aldrich) was added to the medium to inhibit the proliferation of non-neuronal cells. Experiments were performed on DIV5, 6 and 7, in cultures containing <5% astrocytes, as determined by GFAP immunocytochemistry.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from DIV7 primary cortical neurons that had been cultured in high-glucose DMEM in the presence or absence of NMDA, or neurons that had been cultured in MSC-CM in the presence or absence of NMDA, using TRIzol (Invitrogen) according to manufacturers instructions.
|
| Label |
32P
|
| Label protocol |
Total RNA (20μg) was reverse transcribed to [32P]dATP-labeled cDNA. The resulting cDNA probes were hybridized to the Atlas Mouse 1.2 Array II (Clontech #634545) according to the manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
Hybridizations for each experimental condition were repeated twice using independent samples.
|
| Scan protocol |
Membranes were exposed for 4-5 days on a Storm Phosphorimager screen (Molecular Dynamics, Amersham) and read using the Storm Phosphorimager. Hybridization signals were quantified using Image Quant 5.2.
|
| Data processing |
Specific spot intensities were normalized against the average of all spot intensities on each individual array (global normalization) to allow comparisons between independent array experiments.
|
| |
|
| Submission date |
Dec 22, 2010 |
| Last update date |
May 01, 2011 |
| Contact name |
Vivian Tseveleki |
| E-mail(s) |
vtseveleki@hotmail.com
|
| Phone |
+302106478863
|
| Fax |
+302106456547
|
| URL |
http://www.pasteur.gr/205/2074.aspx
|
| Organization name |
Hellenic Pasteur Institute
|
| Department |
Molecular Genetics
|
| Lab |
Molecular Genetics
|
| Street address |
127 Vasilissis Sophias Avenue
|
| City |
Athens |
| ZIP/Postal code |
11521 |
| Country |
Greece |
| |
|
| Platform ID |
GPL145 |
| Series (1) |
| GSE26279 |
Study the effect of mesenchymal stem cells on isolated cortical neurons |
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