|
| Status |
Public on Aug 01, 2018 |
| Title |
Microglia/Macrophages LPS rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Microglia/Macrophages LPS
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6
gender: male
cell type: Adult microglia
treatment: Macrophages, LPS
|
| Treatment protocol |
Adult microglia and bone marrow-derived macrophges were cultured separately for 7 days, as described above; BMDMs were then re-plated on poly-L-lysine coated glass coverslips. Cells were plated at 2x10^5 per 25 mm coverslip (for insertion in 6 well plates) of 4x10^4 per 12 mm coverslip (for insertion in 24 well plates). Coverslips were pre-mounted with 3 small paraffin droplets on the same surface which the cells were plated, as previously described for neuron-astrocyte co-cultures (Jones et al., 2012). BMDMs were allowed to adhere overnight. Bilaminar culture experiments began by inserting coverslips containing BMDM and wax paraffin nodule face down, into culture plates containing adult microglia. All experiments were performed in DMEMF12 serum free media.
|
| Growth protocol |
Adult microglia were re-suspended at 8x10^5 cell-mL (approximately two brains per mL) in media (DMEM F12, 10% FBS, 1% penicillin/streptomycin (P/S)) with 10% L-cell conditioned media, a source of M-CSF (or 10ng/ml recombinant mouse M-CSF [R&D cat no 416-ML-010/CF]), and 50ng/ml recombinant human TGF-β1 (Miltenyi cat no: 130-095-067)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Qiagen Microkit according to manufactors instructions
|
| Label |
biotin
|
| Label protocol |
Sense-strand cDNA was synthesized from 100 ng of total RNA, and fragmentation and labeling were performed to produce ss DNA with the Affymetrix GeneChip® WT Terminal Labeling Kit according to manufacturer’s instructions (ThermoFisher-Affymetrix)
|
| |
|
| Hybridization protocol |
After fragmentation and labeling, 3.5µg DNA target was hybridized on Mouse Clariom™ S Assay (ThermoFisher-Affymetrix) and incubated at 45C in the Genechip® Hybridization oven 640 (ThermoFisher-Affymetrix) for 17 hours at 60 rpm.
|
| Scan protocol |
The microarrays were finally scanned on a GeneChip® scanner 3000 (Affymetrix).
|
| Description |
Adult mouse microglia cultured for 7 days before treatments
|
| Data processing |
Microarray data sets were normalized by the robust multiarray averaging (RMA) method in Affymetrix Expression Console (Affymetrix). To assess whether there were transcripts differentially expressed between activated microglia and activated microglia in the presence of macrophages, normalized data sets were compared in Affymetrix Transcriptome Analysis Console (TAC) Software.
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| |
|
| Submission date |
Aug 10, 2017 |
| Last update date |
Aug 01, 2018 |
| Contact name |
Andrew Greenhalgh |
| E-mail(s) |
adgreenhalgh@gmail.com
|
| Organization name |
The Research Institute of the McGill University Health Center
|
| Street address |
Montreal General Hospital, 1650 Cedar Ave
|
| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3G 1A4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL23038 |
| Series (1) |
| GSE102482 |
Effect of peripherally-derived macrophages in adult microglia (mouse cells) |
|
