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MB Sample ID: SA171012
| Local Sample ID: | LLN_g1 _Intra4_6h |
| Subject ID: | SU001915 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | albino C57BL/6 |
| Age Or Age Range: | 6-8 weeks |
| Gender: | Female |
| Animal Animal Supplier: | Jackson Laboratories (Bar Harbor, ME) |
| Animal Housing: | NCI-Bethesda Animal Facility |
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Collection:
| Collection ID: | CO001908 |
| Collection Summary: | All animal experiments were performed following the guidelines stipulated by the NCI-Bethesda Animal Care and Use Committee. All murine studies were performed using female albino C57BL/6 mice, 6-8 weeks of age, procured from Jackson Laboratories (Bar Harbor, ME). For sub-cutaneous tumor studies, 6x10^6 cells of stably transduced CT2A glioma cells with mCherry-firefly luciferase were injected in 100 μL PBS. For intracranial tumor studies, 1x10^2 CT2A cells with mCherry- firefly luciferase were injected in 2uL PBS. GSK126 for in vivo studies was obtained from the NCI- Drug Synthesis and Chemistry Branch and dissolved in 20% SBE-Cyclodextrin (MedChemExpress, HY-17031) pH 4-4.5 with 1N acetic acid. Vehicle was 20% SBE-Cyclodextrin pH 4-4.5 with 1N acetic acid. Water-soluble dexamethasone (Sigma Aldrich; D2915) was administered at 1mg/kg/day also by intraperitoneal injection. Anti PD-1(InVivoMAb; BE0146) or isotype control, rat IgG2a (InVivoMAb; BE0089) were also injected intraperitoneally. Subcutaneous tumor growth was measured using calipers and thereafter tumor volume was calculated using the formula for the volume of an ellipsoid given below. In the case of intracranial tumors, tumor growth was measured using the luminescence reader IVIS Ilumina and analyzed using LivingImage Software. Samples were collected from mice with three biological replicates. Serum (50 µL) was transferred to 200 μL ice-chilled (4°C) MilliQ H2O. Tissue (~16 mg) was measured from subcutaneous and intracranial samples followed by addition of 250 μL MilliQ H2O. Then, samples were sonicated at 40 amps (~30 s) until homogeneous. 80 μL of at 0.150 µg/mL debrisoquine in 60% methanol (MeOH)/40% water(aq) reagent was added. 500 μL chilled (-20°C) MeOH was added, vortexed (med) and incubated 15 min on ice. 250 μL chilled (-20°C) Chloroform was added, vortexed (high) and incubated 20 min in ice on rotating mixer. Mixture was centrifuged (13,000x g) for 18 min at 4°C. 705 μL of hydrophilic upper layer was aspirated and transferred to separate 1.5 mL microtubes, dried to completion under N2 gas sample concentrator, and stored at -80°C until LC/MS quantification of GSK126. |
| Sample Type: | Tumor cells |
| Collection Method: | Tumor Tissue/Serum Extract |
| Collection Location: | Intracranial/Subcutaneous/Serum from Mice |
| Collection Frequency: | Timepoints |
| Collection Duration: | 0, 2h, 6h and 10h |
| Volumeoramount Collected: | Serum (50 µL)/Tumor (~16 mg) |
| Storage Conditions: | -80℃ |
| Collection Vials: | 15 mL |
| Storage Vials: | 1.7 mL |
| Collection Tube Temp: | On Ice |
| Additives: | Extraction reagent |
