|
| Status |
Public on Feb 17, 2015 |
| Title |
HuMonocytes_Medium_1hr_LPS_2hr_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Human monocytes, pre-incubated in medium for 1 hr, stimulated with 1ng/ml LPS for 2 hrs
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human peripheral blood-derived monocytes
agent: pre-incubated in medium for 1 hr, stimulated with 1ng/ml LPS for 2 hrs
|
| Treatment protocol |
To study the pro-inflammatory activities of Cpn60.1, human monocytes were purified from three healthy donors and incubated with 10 μg/ml M. tuberculosis Cpn60.1 (or 100 ng/ml LPS as a positive control or medium only as a negative control). To study the inhibitory effectds of Cpn60.1 on human monocytes, the cells were pre-exposed to 10μg/ml or 0.1ng/ml of Cpn60.1, washed and incubated with 1ng/ml LPS for further 2 hours. For positive control, the cells were pre-exposed to medium before washing and LPS stimulation while for the negative control, the cells were pre-exposed to medium, washed and again exposed to medium alone.
|
| Growth protocol |
Human monocytes were purified from peripheral blood by negative selection and maintained in RPMI medium containing 2% autologous plasma before stimuation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from three healthy donors using the RNEasy Kit according to the manufacturer’s instructions (Qiagen)
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA was prepared according to the standard Affymetrix protocol described in GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual v.4
|
| |
|
| Hybridization protocol |
Biotinylated cDNA was addded to the hybridization cocktail, and incubated on Human GeneChip ST Array for 17 hours, at 60rpm at 46°C. Staining and washing was done on Fluidics station 450.
|
| Scan protocol |
Human GeneChip arrays were scanned on GeneChip Scanner 3000 7G.
|
| Description |
Study on inhibitory activity of M. tuberculosis Cpn60.1 on activated human monocytes
|
| Data processing |
The arrays were normalized with Robust Multi-Chip Average algorithm (RMA) using the Affymetrix Expression Console 1.1. Probe sets with signals below the 85% percentile across all arrays or without gene annotation were removed from the data set leaving 17,649 probe sets for further analysis. The statistical analysis of samples (comparing triplicate biological replicates of treated to untreated cells) was performed using a moderated t-test in Limma using a p-value ≤ 0.05 adjusted for multiple testing, and a minimum fold change of 1.5.
|
| |
|
| Submission date |
Oct 29, 2009 |
| Last update date |
Feb 17, 2015 |
| Contact name |
Ana Cehovin |
| E-mail(s) |
[email protected]
|
| Phone |
02075943094
|
| Fax |
02075943095
|
| Organization name |
Imperial College London
|
| Department |
Centre for Molecular Microbiology and Infection
|
| Lab |
Pelicic Lab
|
| Street address |
Exhibition Road
|
| City |
London |
| State/province |
United Kingdom |
| ZIP/Postal code |
SW7 2AZ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE18794 |
Mycobacterium tuberculosis Chaperonin 60.1 has Bipolar Effects on Human peripheral blood-derived Monocytes |
|
