|
| Status |
Public on Apr 30, 2024 |
| Title |
APP_WT_vs_WT_R3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: differentiated NPCs from wild-type embryo telencephalon
|
| Treatment protocol |
For neural differentiation, neurospheres were washed twice with DMEM/F12 medium following centrifugation for 3 minutes at 100xG and then were allowed to attach to poly-L-lysine and laminin precoated plastic flasks and cultured with DMEM/F-12, supplemented with 2% B-27, in the absence of FGF-2 and EGF. Progenitor cells were differentiated for 7 days in the incubator at 37°C with controlled humidity and 5% CO2. Neurospheres from APP/PS1 mice were submitted to different drug treatments on days 0, 3, and 6 of the differentiation phase. APP_WT neurospheres were maintained untreated; c neurospheres were treated with 1µM BK (Tocris Bioscience) and HOE_APP neurospheres were treated with 1µM HOE-140 (Tocris Bioscience). The drug concentrations were defined based on Pillat et al. 2016 (Pillat et al. 2016). On the seventh day of differentiation, RNAs from neurospheres were extracted to perform the microarray analysis.
|
| Growth protocol |
NPCs were isolated from embryo telencephalon (day 13) of APPswe/PSldE9 C57BL/6 mice. All animal experiments were performed according to protocols approved by the ethics committee (authorization number: 10/2013). Isolated telencephalons were mechanically and enzymatically dissociated. A concentration of 2×105 cells/ml was seeded in 2% B-27 (Life Technologies, Carlsbad, CA), Dulbecco's modified Eagle's medium (DMEM) with Ham's F-12, antibiotics (100 units/ml penicillin and 100 g/ml streptomycin), 20 ng/ml EGF and 20 ng/ml FGF2 (Sigma-Aldrich, St Louis, MO) and cultured at 37 °C in a water-saturated atmosphere with 5% of CO2. Cultures were grown for 5 days until formation of neurospheres was observed. Neurospheres were washed twice with DMEM/F12 at 300 r.p.m for 3 min.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from BK and HOE treated, and untreated APPswe/PS1dE9 neurospheres was extracted using the RNAspin Mini Kit (GE Healthcare, UK), according to the manufacturer instructions. The RNA was treated with DNAse.
|
| Label |
Cy3
|
| Label protocol |
100 ng of total RNA were DNAse-treated and amplified and labeled using Agilent Low RNA Input Fluorescent Linear Amplification Kit following manufacturer's instructions.
|
| |
|
| Channel 2 |
| Source name |
APPswe/PS1dE9 cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: differentiated NPCs from APPswe/PS1dE9 embryo telencephalon
|
| Treatment protocol |
For neural differentiation, neurospheres were washed twice with DMEM/F12 medium following centrifugation for 3 minutes at 100xG and then were allowed to attach to poly-L-lysine and laminin precoated plastic flasks and cultured with DMEM/F-12, supplemented with 2% B-27, in the absence of FGF-2 and EGF. Progenitor cells were differentiated for 7 days in the incubator at 37°C with controlled humidity and 5% CO2. Neurospheres from APP/PS1 mice were submitted to different drug treatments on days 0, 3, and 6 of the differentiation phase. APP_WT neurospheres were maintained untreated; c neurospheres were treated with 1µM BK (Tocris Bioscience) and HOE_APP neurospheres were treated with 1µM HOE-140 (Tocris Bioscience). The drug concentrations were defined based on Pillat et al. 2016 (Pillat et al. 2016). On the seventh day of differentiation, RNAs from neurospheres were extracted to perform the microarray analysis.
|
| Growth protocol |
NPCs were isolated from embryo telencephalon (day 13) of APPswe/PSldE9 C57BL/6 mice. All animal experiments were performed according to protocols approved by the ethics committee (authorization number: 10/2013). Isolated telencephalons were mechanically and enzymatically dissociated. A concentration of 2×105 cells/ml was seeded in 2% B-27 (Life Technologies, Carlsbad, CA), Dulbecco's modified Eagle's medium (DMEM) with Ham's F-12, antibiotics (100 units/ml penicillin and 100 g/ml streptomycin), 20 ng/ml EGF and 20 ng/ml FGF2 (Sigma-Aldrich, St Louis, MO) and cultured at 37 °C in a water-saturated atmosphere with 5% of CO2. Cultures were grown for 5 days until formation of neurospheres was observed. Neurospheres were washed twice with DMEM/F12 at 300 r.p.m for 3 min.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from BK and HOE treated, and untreated APPswe/PS1dE9 neurospheres was extracted using the RNAspin Mini Kit (GE Healthcare, UK), according to the manufacturer instructions. The RNA was treated with DNAse.
|
| Label |
Cy5
|
| Label protocol |
100 ng of total RNA were DNAse-treated and amplified and labeled using Agilent Low RNA Input Fluorescent Linear Amplification Kit following manufacturer's instructions.
|
| |
|
| |
| Hybridization protocol |
Agilent Gene Expression Hybridization Kit were used and labeled targets were incubated to na Agilent SurePrint G3 Mouse GE 8x60K Microarray enclosed in Agilent Microarray Hybridization Chamber. After hybridization, slides were washed sequentially.
|
| Scan protocol |
Agilent Feature Extraction Software (v 9.5)
|
| Description |
Replicate 3 of 4: APPswe/PS1dE9 mutant vs. WT NPCs
|
| Data processing |
Four biological replicates were assessed with dye swap. gProcessedSignal and rProcessedSignal values were used to calculate log2 BK_APP/APP_WT ratios and log2 HOE_APP/APP_WT. Only the probes with significantly greater expression (as determined via the 2-sided t-test) compared with the corresponding background level (flag ‘IsWellAboveBG’) in 3 of the 4 control sample replicates were defined as expressed and further analysed.
|
| |
|
| Submission date |
Nov 01, 2023 |
| Last update date |
Apr 30, 2024 |
| Contact name |
Eduardo Moraes Reis |
| E-mail(s) |
emreis@iq.usp.br
|
| Phone |
+55-11-30912173
|
| Organization name |
University of São Paulo
|
| Department |
Biochemistry
|
| Street address |
Av. Prof. Lineu Prestes, 748
|
| City |
São Paulo |
| State/province |
SP |
| ZIP/Postal code |
05508-900 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL13912 |
| Series (1) |
| GSE246792 |
Bradykinin promotes immune responses in differentiated embryonic neurospheres carrying APP swe and PS1 dE9 mutations |
|
