|
| Status |
Public on Aug 09, 2019 |
| Title |
MPLA_4: BMDM_treated with MPLA as M1 standards |
| Sample type |
RNA |
| |
|
| Source name |
Bone Marrow Derrived Macrophages
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Bone Marrow Derrived Macrophages
cytokine exposure: MPLA
time of cytokine exposure: 4 days
nanoparticle treatment: N/A
|
| Treatment protocol |
At day 7 ex vivo, culture media was replaced with macrophage conditioned media (macrophage complete medium supplemented with 20 ng/mL MPLA (Sigma, cat#L6895) or 20 ng/mL IL4 (eBioscience, cat# 34-8041)). BMDMs were used between 7 - 21 days ex vivo for all experiments. For nanoparticle treatment, 1 microgram of either IRF5-NPs or eGFP-NPs were co-incubated with BMDMs for 1 hours, followed with completly removal of nanoparticles and 2X wash with culture medium.
|
| Growth protocol |
To prepare BMDMs, bone marrow progenitor cells were harvested from mouse bone marrow cavity of femur following established protocol [Curr Protoc Immunol. 2008 Nov;Chapter 14:Unit 14.1.]. These cells were then seeded in 100 mm culture dishes in complete media (DMEM supplemented with 4.5 g/L D-glucose, L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL, Glutamax 50 mL/500 mL, supplemented with 20 ng/mL M-CSF (Peprotech, cat#315-02) at a seeding density of 0.5 – 1.0 e6/ml. Cells were allowed to grow ex vivo under 37 °C, 5% CO2 for 7 days to fully derive into BMDMs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using RNeasy Plus Universal Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Sample RNA was quantified using a NanoDrop Microvolume Spectrophotometer (Thermo Fisher, Waltham, MA) and quality checked and RINe scores determined by TapeStation (Agilent, Santa Clara, CA).
|
| Label |
N/A
|
| Label protocol |
Ligation master mix (provided by NanoString) was added to the mRNA solution and incubated under 48 °C for 5min to allow ligation, followed with purification to remove unligated molecules.
|
| |
|
| Hybridization protocol |
The ligated RNA were co-incubated with miRNA capture probeset, miRNA reporter codeset in hybridization buffer following the protocol provided by NanoString. The hybridation happened in 65 °C for at least 12 hours.
|
| Scan protocol |
All samples were read on nCounter Analysis System (NanoString Technologies).
|
| Data processing |
Raw data was processed and checked for quality using the R/Bioconductor package NanoStringQCPro (Nickles D, Sandmann T, Ziman R and Bourgon R (2017). NanoStringQCPro: Quality metrics and data processing methods for NanoString mRNA gene expression data. R package version 1.10.0.). Expression values were normalized to the geometric mean of housekeeping genes and log2-transformed using nSolver 4.0 software (NanoString Technologies). FDR for ratio data was calculated from the p-values returned by the t-test using the Benjamini-Yekutieli method.
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| |
|
| Submission date |
Sep 20, 2018 |
| Last update date |
Aug 09, 2019 |
| Contact name |
Matthias Stephan |
| Organization name |
Fred Hutchinson Cancer Research Institute
|
| Street address |
1100 Fairview Ave. N., Thomas Research Buildin
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109-9024 |
| Country |
USA |
| |
|
| Platform ID |
GPL25266 |
| Series (1) |
| GSE120254 |
Identify signature genes associated with nanoparticle carrying IRF5 mRNA treatment in bone marrow derrived macrophages |
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