|
| Status |
Public on Jun 03, 2014 |
| Title |
BMDM, Tpl2 KO, unstimulated, biological replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Bone marrow derive macrophage
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: Tpl2 deficient
treatment: unstimulated
|
| Treatment protocol |
For individual experiments, murine BMDMs or adherence purified PECs were plated at either 0.5 x 106/mL or 1 x 106/mL in supplemented DMEM medium. Cells were left unstimulated (as a control) or stimulated with lipopolysaccharide (LPS) from Escherichia coli (0111:B4, 1 μg/mL) for various time-points at 37°C, 5% CO2. For inhibition studies, BMDMs were pre-treated with the following inhibitors in supplemented DMEM medium for 30 min at 37°C, 5% CO2 prior to stimulation with LPS: LY-294,002 hydrochloride, 20 μM (Sigma); Rapamycin, 30 nM (Sigma); U0126 ethanolate, 20 μM (Sigma).
|
| Growth protocol |
BM was harvested from tibiae and femurs of mice by flushing with supplemented DMEM using a 10 mL syringe and 25-gauge needle. Cells were disaggregated by gentle pipetting and centrifuged at 1200 rpm for 10 min at RT. The cells were then re-suspended in ACK lysing buffer (Invitrogen) for 30 sec to lyse red blood cells. Cells were washed in PBS (20-30 mL) and centrifuged again at 1200 rpm for 10 min. Cell pellet was resuspended in supplemented DMEM for cell counting. Differentiated macrophages were obtained by culturing BM cells on sterile petri dishes at 2 x 106/mL in supplemented DMEM with the addition of macrophage colony-stimulating factor (M-CSF; 10 ng/mL) for 7-10 days at 37°C, 5% CO2. Cells were adherence purified on day 7-10 by removing media, washing adherent cells with PBS and then harvesting by incubating in 10 mL cell dissociation buffer (Invitrogen) for 15 min at 37°C. Plates were washed with PBS to collect dislodged cells, and cells were centrifuged at 1200 rpm for 10 min. Cell pellets were resuspended in supplemented DMEM for cell counting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was extracted using an RNeasy Mini kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Approximately 10 ug of total RNA was labeled using MessageAmp™ II-Biotin Enhanced Kit (Ambion)
|
| |
|
| Hybridization protocol |
Approximately 10 ug of biotinylated cRNA was hybridized to GeneChip Mouse Genome 430 2.0 arrays (Affymetrix) at 45 degree for 16 hours in accordance with the manufacturer’s protocols
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
|
| Description |
Gene expression data from BMDM
|
| Data processing |
Expression values were determined with GeneChip Operating Software (GCOS) v1.1.1 software. All data analysis was performed using GeneSpring software GX 11.0. Expression values for each probe were normalized using the Robust Multichip Average (RMA) method.
|
| |
|
| Submission date |
Jun 26, 2013 |
| Last update date |
Jun 03, 2014 |
| Contact name |
Lai Wei |
| E-mail(s) |
weil2@mail.nih.gov
|
| Phone |
3014961480
|
| Organization name |
NIH/NEI/NCCAM
|
| Street address |
10 Center Dr. Room 2B47
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE48338 |
Tpl2 promotes chemokine/chemokine receptor expression and macrophage migration during acute inflammation |
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