|
| Status |
Public on Jan 10, 2006 |
| Title |
Pac-1-/- TG-macrophages_LPS 6h |
| Sample type |
RNA |
| |
|
| Source name |
peritoneal lavage
|
| Organism |
Mus musculus |
| Characteristics |
Thioglycollate-elicited (72 h) peritoneal macrophages from PAC-1 deficient mice stimulated with 100 ng/ml of LPS for 6 hours
|
| Treatment protocol |
Pac1–/– mice were injected intraperitoneally with 2 mL of 3% thioglycollate (Sigma) 4 days prior to sacrifice. Peritoneal macrophages were collected by lavaging the peritoneal cavity with 10 ml of sterile RPMI 1640 medium. Macrophages were purified by adherence to tissue culture plates for 2 h. The macrophage population was approximately 92-95% mac-1 positive as assessed by PE-labelled mac-1 antibodies (BD Pharmingen). Morphology was assessed by cytospin and staining with giemsa. Peritoneal macrophages at 2 × 10^6 cells/mL were treated with 100 ng/ml of LPS (Sigma, St Louis Lot # 072K4096) for 6 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol was used for total RNA extraction. cRNA was prepared according to methods for small RNA samples (Baugh et al, Nucleic acids research 29:E29, 2001). 500-1000 ng of RNA was reverse transcribed to cDNA using a poly(T) primer containing a T7-(dT)24 (Geneworks, Australia).
|
| Label |
PE
|
| Label protocol |
cRNA was transcribed from cDNA and biotinylated using the BioArray High Yield RNA Transcript Labelling Kit (Enzo Diagnostics, Farmingdale, NY).
|
| |
|
| Hybridization protocol |
Fifteen micrograms of cRNA was fragmented by heating at 94°C for 35 min in fragmentation buffer (40mM Tris-acetate (pH 8.1), 125mM KOAc, 30mM MgOAc). Hybridization cocktails were then made by adding fragmented cRNA, control cRNAs, grid alignment oligonucleotides and blocking reagents. These mixtures were hybridized for 16 hours to individual U133A and B genechips (Affymetrix, Santa Clara, CA) at 45 °C. Washing and staining of the hybridized arrays were performed using an Affymetrix Fluidics Station.
|
| Description |
Pac-1-/- thioglycollate-elicited macrophages stimulated with LPS for 6 hours
|
| Data processing |
MAS5.0
|
| |
|
| Submission date |
Jan 06, 2006 |
| Last update date |
Jan 09, 2006 |
| Contact name |
Charles Reay Mackay |
| E-mail(s) |
c.mackay@garvan.org.au
|
| Phone |
+61-2-92958405
|
| Fax |
+61-2-92958404
|
| Organization name |
Garvan Institute for Medical Research
|
| Street address |
384 Victoria St
|
| City |
Darlinghurst |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
| |
|
| Platform ID |
GPL32 |
| Series (2) |
| GSE3993 |
Pac1+/+ versus Pac1-/- TG-macrophages_LPS 6h |
| GSE4014 |
Pac1+/+ versus Pac1-/- in macrophages and mast cells |
|
