Return to study ST001838 main page
MB Sample ID: SA170982
| Local Sample ID: | SER_g1 _Subcutaneous8_10h |
| 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 |
Select appropriate tab below to view additional metadata details:
Subject:
| 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 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| SER_g1 _Subcutaneous8_10h | SA170982 | FL020594 | GSK | Treatment |
| SER_g1 _Subcutaneous8_10h | SA170982 | FL020594 | 10.0 | Gsk Exposure Time (h) |
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 |
Treatment:
| Treatment ID: | TR001928 |
| Treatment Summary: | Extracts of Gracilaria edulis were prepared through two different approaches, namely, sequential and direct, following the procedure of Subermaniam et al. (2020). For the sequential process, the solvents were used in the order of increasing polarity viz. ethyl acetate < acetone. For the direct extracts, ethyl acetate and acetone were used separately. |
Sample Preparation:
| Sampleprep ID: | SP001921 |
| Sampleprep Summary: | 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 |
| Sampleprep Protocol ID: | Extraction |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | LCMS Extraction |
| Extract Cleanup: | Bligh-Dyer biphasic separation, discard protein disk and dried to completion under N2 gas |
| Sample Resuspension: | 80 uL 60% MeOH (aq) |
| Sample Spiking: | Internal standard 0.150 µg/mL debrisoquine(IS) was added to each |
Combined analysis:
| Analysis ID | AN002980 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Agilent 1290 Infinity II |
| Column | Agilent AdvanceBio Glycan Map 2.1 x 100 mm 2.7µm column |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Agilent 6545 |
| Ion Mode | POSITIVE |
| Units | ng |
Chromatography:
| Chromatography ID: | CH002209 |
| Chromatography Summary: | Samples were injected at 8 µL over an 8.3 min gradient on the AdvanceBio Glycan Map 2.1 x 100 mm 2.7µm column at 35°C with a flow rate of 0.220 mL/min. The LC gradient only utilized LC/MS grade reagents when preparing mobile phases, A (88:12 H2O/acetonitrile (ACN) and B 90% ACN (aq). Both mobile phases were composed with 10 mM ammonium acetate and titrated to pH 6.85 using formic acid and ammonium hydroxide. The LC gradient was initially 100% B for 0.25 min and then ramped to 55% B at 2.5 min; 49% B at 4.5 min; 35% B at 5.5 min; 20% B at 6 min; held for 0.5 min; 15% B at 7 min; 100%B at 8.3 min followed by equilibration for 1.2 min. |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Agilent AdvanceBio Glycan Map 2.1 x 100 mm 2.7µm column |
| Column Pressure: | 600 bar |
| Column Temperature: | 35°C |
| Flow Gradient: | The LC gradient was initially 100% B for 0.25 min and then ramped to 55% B at 2.5 min; 49% B at 4.5 min; 35% B at 5.5 min; 20% B at 6 min; held for 0.5 min; 15% B at 7 min; 100%B at 8.3 min followed by equilibration for 1.2 min. |
| Flow Rate: | 0.220 mL/min |
| Injection Temperature: | 4°C |
| Internal Standard: | Continuous accurate mass correction was achieved by infusing proprietary Agilent Technologies API-TOF reference mass standard solution |
| Retention Time: | 2.5-2.7 min |
| Sample Injection: | 8 uL |
| Sampling Cone: | 2kV |
| Solvent A: | 88% water/12% acetonitrile; 10 mM ammonium acetate, pH 6.85 |
| Solvent B: | 90% acetonitrile/10% water; 10 mM ammonium acetate, pH 6.85 |
| Analytical Time: | 8.3 min |
| Capillary Voltage: | 3 kV |
| Sheath Liquid: | N2 |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002770 |
| Analysis ID: | AN002980 |
| Instrument Name: | Agilent 6545 |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | MS/MS Using Masshunter Qtof Quant-My-Way 10.0 software, GSK126 was detected at elution time 2.6 min using precursor ion m/z 527.3129 and transition m/z 375.2183 generated via N2 gas collision-induced fragmentation (CID) at a collision energy (CE) of 12 V. Internal standard (IS) debrisoquine detected at elution time of 2.5 mins with precursor m/z 176.1182 and transition m/z 134.0964 generated at CE 12V. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 325°C |
| Capillary Voltage: | 3kV |
| Collision Energy: | 12V |
| Collision Gas: | N2 |
| Dry Gas Flow: | 9 L/min |
| Dry Gas Temp: | 250°C |
| Fragment Voltage: | 100 V |
| Fragmentation Method: | MS/MS |
| Ion Source Temperature: | 325°C |
| Mass Accuracy: | 2 kDa |
| Source Temperature: | 325°C |
| Cdl Temperature: | RT |
| Desolvation Temperature: | 325°C |
| Nebulizer: | 45 psig |
| Octpole Voltage: | 750V |
