|
| Status |
Public on Oct 30, 2024 |
| Title |
LPS_activated_THP-1_macrophages_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
THP-1 cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: acute monocytic leukemia
treatment: LPS_activated_THP-1_macrophages
|
| Treatment protocol |
The cells were rinsed 3 × times using the medium followed by treatment with either LPS (100 ng/ml) for control cells or LPS (100 ng/ml) and Fh-ES (7 µg/ml) for experimental cells. Fh-ES concentration was used to stimulate THP-1 macrophages, resulting in an enrichment of the medium with endotoxin to a final concentration of 0.06 EU/ml, what fills the strict FDA recommendation for endotoxin level even for solutions that have a contact with cerebrospinal fluid
|
| Growth protocol |
The monocytes were cultured at 37°C with 5% CO2 in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Once the cells reached the appropriate number, they were seeded at 5E5 cells per a well in a 24-well cell culture plate and differentiated into macrophages for 48 hours using PMA (30 ng/ml).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA, which also included miRNA, was isolated using miRNeasy Mini Kit (Qiagen) according to the manufacturer’s procedure. RNA quality was determined using Agilent 2100 Bioanalyzer (USA) and RNA 6000 Nano Kit (Agilent, Germany). All RNA samples taken for microarray analysis were of high quality (RIN was between 9.5 and 10.0).
|
| Label |
Cy-3
|
| Label protocol |
One hundred ng of total RNA with miRNA Spike-In (microRNA Spike-In Kit, Agilent Technologies) added were taken for labeling reaction. miRNA was labeled with Cyanine 3-pCp using the Complete Labeling and Hyb Kit (Agilent Technologies) according to the manufacturer’s procedure. The labeled RNA samples were purified on Micro Bio-Spin 6 columns to remove DMSO and unbound Cyanine 3-pCp.
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| |
|
| Hybridization protocol |
Each Cyanine-3-labeled RNA samples were hybridized onto microarray for 20h at 55°C in hybridization rotating oven (Agilent Technologies).
|
| Scan protocol |
Acquisition and analysis of hybridization intensities were performed using Agilent DNA Microarray Scanner G2505C. Images were quantified using Agilent Feature Extraction Software (version 10.7.3.1).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (version 10.10.1.1) using default parameters (protocol miRNA_1010_Sep10 and Grid: 046066_D_F_20141006) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Sep 13, 2024 |
| Last update date |
Oct 30, 2024 |
| Contact name |
Piotr Bąska |
| E-mail(s) |
[email protected]
|
| Phone |
48225936038
|
| Organization name |
Warsaw University of Life Sciences WULS – SGGW
|
| Department |
Department of Preclinical Sciences
|
| Lab |
Division of Pharmacology and Toxicology
|
| Street address |
Ciszewskiego 8
|
| City |
Warsaw |
| State/province |
Mazowieckie |
| ZIP/Postal code |
02-786 |
| Country |
Poland |
| |
|
| Platform ID |
GPL21575 |
| Series (1) |
| GSE277108 |
Analysis of miRNA expression upon stimulation of LPS activated THP-1 macrophages with Fasciola hepatica Excretory-Secretory Products (Fh-ES) |
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