|
| Status |
Public on Dec 19, 2009 |
| Title |
Interstitial leukocytes from Rag1 KO, NK-depleted mice exposed to silica, replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RagKO, NK-deficient lung interstitial leukocytes
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Rag1-/-
tissue: lung
cell type: interstitial leukocytes
age: 6-8 weeks
treatment: silica
|
| Treatment protocol |
For “chronic” exposure studies, Rag1-/- NK-depleted and C57Bl/6 wild-type mice were anesthetized with ketamine (80 mg/kg) and instilled i.n. with either 25 μl of sterile saline, 1 mg crystalline silica (SiO2) or 0.5 mg titanium dioxide (TiO2) suspended in 25 μl of sterile saline once a week for four weeks.
|
| Growth protocol |
Rag1-/- mice were injected i.p. with 300 μg of anti-NK1.1 mAb on days -6, -3, and -1 prior to the first i.n. instillation (day 0). NK cell depletion was maintained via weekly injections of 300 μg of anti-NK1.1 mAb throughout the course of the study, which was administered two days prior to particle exposure.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The lungs were asceptically removed, cut away from the heart, and placed in ice cold sterile phosphate-buffered saline (PBS, pH 7.4). Thereafter, the lungs were minced into small pieces and incubated in complete RPMI 1640 culture medium supplemented with 10% FCS, antibiotic/antimycotic solution, β-mercaptoethanol, and sodium pyruvate containing 1 mg/ml collagenase IA with 1 µl DNAase I during 2 hours at 37oC. Digested lungs were further disrupted by gently pushing the tissue through a 70 μm cell strainer. Enzymatic action was terminated by adding excess RPMI medium and pelleting by centrifugation at 1500 rpm for 5 minutes at 4oC. Leukocytes were isolated by centrifugation over a 40-70% Percol gradient. Total RNA was isolated using Trizol according to manufacturer's protocol, followed by Qiagen RNeasy RNA cleanup with DNase digest.
|
| Label |
Cy5
|
| Label protocol |
Agilent Quick-Amp Labeling Kit, Dual Color.
|
| |
|
| Channel 2 |
| Source name |
Universal Mouse Reference RNA
|
| Organism |
Mus musculus |
| Characteristics |
supplier: Stratagene
|
| Treatment protocol |
For “chronic” exposure studies, Rag1-/- NK-depleted and C57Bl/6 wild-type mice were anesthetized with ketamine (80 mg/kg) and instilled i.n. with either 25 μl of sterile saline, 1 mg crystalline silica (SiO2) or 0.5 mg titanium dioxide (TiO2) suspended in 25 μl of sterile saline once a week for four weeks.
|
| Growth protocol |
Rag1-/- mice were injected i.p. with 300 μg of anti-NK1.1 mAb on days -6, -3, and -1 prior to the first i.n. instillation (day 0). NK cell depletion was maintained via weekly injections of 300 μg of anti-NK1.1 mAb throughout the course of the study, which was administered two days prior to particle exposure.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The lungs were asceptically removed, cut away from the heart, and placed in ice cold sterile phosphate-buffered saline (PBS, pH 7.4). Thereafter, the lungs were minced into small pieces and incubated in complete RPMI 1640 culture medium supplemented with 10% FCS, antibiotic/antimycotic solution, β-mercaptoethanol, and sodium pyruvate containing 1 mg/ml collagenase IA with 1 µl DNAase I during 2 hours at 37oC. Digested lungs were further disrupted by gently pushing the tissue through a 70 μm cell strainer. Enzymatic action was terminated by adding excess RPMI medium and pelleting by centrifugation at 1500 rpm for 5 minutes at 4oC. Leukocytes were isolated by centrifugation over a 40-70% Percol gradient. Total RNA was isolated using Trizol according to manufacturer's protocol, followed by Qiagen RNeasy RNA cleanup with DNase digest.
|
| Label |
Cy3
|
| Label protocol |
Agilent Quick-Amp Labeling Kit, Dual Color.
|
| |
|
| |
| Hybridization protocol |
Agilent Gene Expression Hybridization Kit.
|
| Scan protocol |
Axon GenePix 4000B scanner, Axon GenePix Pro 5.0 software.
|
| Description |
Biological replicate 2 of 2, RagKO silica.
RagKO-si-2
|
| Data processing |
Axon GenePix Pro 5.0, normalization using a LOWESS R script written by Terry Speed, Microsoft Excel. Matrix data was filtered to include only non-control genes and genes present above background in at least 2 of 4 replicates for wild-type or RagKO data.
|
| |
|
| Submission date |
Dec 18, 2009 |
| Last update date |
Dec 18, 2009 |
| Contact name |
Corbin Schwanke |
| E-mail(s) |
corbin.schwanke@umontana.edu
|
| Phone |
406-243-4571
|
| Fax |
406 243-4571
|
| Organization name |
University of Montana
|
| Department |
Biomedical and Pharmaceutical Sciences
|
| Lab |
The Center for Environmental Health Sciences
|
| Street address |
|
| City |
Missoula |
| State/province |
MT |
| ZIP/Postal code |
59801 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE19563 |
Gene expression response to silica treatment in wild-type C57 black 6 mice vs Rag1 knockout, NK-depleted mice |
|
