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MB Sample ID: SA194288

Local Sample ID:KL_3
Subject ID:SU002148
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57Bl/6 GEMMs
Age Or Age Range:Mutants: K: 90 days, KK: 60 days, KL and KKL: 40 days post Ad5-CMV-Cre
Gender:Male and female

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Subject:

Subject ID:SU002148
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57Bl/6 GEMMs
Age Or Age Range:Mutants: K: 90 days, KK: 60 days, KL and KKL: 40 days post Ad5-CMV-Cre
Gender:Male and female

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
KL_3SA194288FL023820Kras/Lkb1treatment

Collection:

Collection ID:CO002141
Collection Summary:At defined timepoints (K: 90 days, KK: 60 days, KL and KKL: 40 days post Ad5-CMV-Cre), mice were sacrificed by cardiac puncture and lungs and plasma harvested for downstream analysis.
Sample Type:Lung
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002160
Treatment Summary:Mice were genotyped using primers outlined in the Key Resources Table. Seven-to-eight-week old conditional mice were intranasally (i.n) infected with 20 ul of 1x1010 PFU/ml Ad5-CMV-Cre virus (University of Iowa Gene Transfer Core Facility) according to standard procedures62. For anti-PD1 survival study, 20 days following Ad5-CMV-Cre, KL and KKL mice were randomly assigned to anti-PD1 or isotype control antibody (200 g RMP1-14 or Rat IgG2a; BioXCell) treatment arms (n = 6/arm). Each cycle (every 20 days) consisted of intra-peritoneal injection (200 l into left and right flank) three times over 6 days (day 0, 3, 6). Mice were collected at ethical endpoint for Kaplan Meier survival analysis. For combination anti-PD1/CB-839 study, 20 days following Ad5-CMV-Cre, KL mice were randomly assigned to vehicle/isotype, vehicle/anti-PD1, CB-839/isotype or CB-839/anti-PD1 treatment arms (study 1: n = 6/arm; study 2: n = 4/arm). On day 20, mice received one cycle of anti-PD1 or isotype control (as above) and 200 mg/kg CB-839 (2 % w/v CB-839; BLDpharm, in vehicle) or vehicle (20 % Solutol HS15 / 20 % SB-β-CD (Captisol) / 50 mM phosphate buffer, pH 7.4) twice daily by oral gavage until collection on day 29, when mice were collected 12 hours following the final dose. At harvest, mice were sacrificed by cardiac bleed and the left lung lobe was weighed and processed for flow cytometry with spleens, while right lung lobes were inflated with 4 % paraformaldehyde and processed for histology.
Treatment Compound:200 ug RMP1-14 or Rat IgG2a; Combination treatments: vehicle/isotype, vehicle/anti-PD1, CB-839/isotype or CB-839/anti-PD1 treatment arms
Treatment Route:by oral gavage
Treatment Dose:multiple dose 200 ug. Combination treatments: 200 mg/kg CB-839 (2 % w/v CB-839; BLDpharm, in vehicle) or vehicle (20 % Solutol HS15 / 20 % SB-β-CD (Captisol) / 50 mM phosphate buffer, pH 7.4) twice daily by oral gavage until collection on day 29,

Sample Preparation:

Sampleprep ID:SP002154
Sampleprep Summary:Tumor tissue was flash frozen and crushed using a mortar and pestle in liquid nitrogen. To the Eppendorf tubes 150 µL of cold 80 % acetonitrile was added and vortexed quickly. The suspension was transferred to a fresh Eppendorf tube. Another 150 µL portion of extraction solvent was added to the tubes, vortexed and the suspension combined with the first portion. To the combined extraction mixture 75 µL of CHCl3 was added and samples were mixed for 1 h in a cool room to ensure complete extraction. The samples were centrifuged at 4 °C at 14.8 g for 10 min, supernatant transferred to new tubes and evaporated at 20 °C under a stream of nitrogen. BSA assay was performed on the protein pellet by dissolving all protein in equal volume of detergent solution (4 % v/v SDS in water). Dried samples were resolubilized in appropriate volume of CHCl3 : MeOH : H2O (1 : 3 :1, v/v) mixture based on measured protein content, shaken for 15 min at room temperature, centrifuged at 4 °C at 14.8 g for 10 min. 100 µL of the supernatant was transferred to LCMS vials. 10 µL injected for metabolomics analysis.
Processing Storage Conditions:-80℃
Extract Storage:-80℃

Combined analysis:

Analysis ID AN003365 AN003366
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS
Column SeQuant ZIC-pHILIC (150 x 4.2mm,5um) SeQuant ZIC-pHILIC (150 x 4.2mm,5um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units

Chromatography:

Chromatography ID:CH002489
Chromatography Summary:LCMS data was acquired on Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) coupled with high-performance liquid chromatography (HPLC) system Dionex Ultimate® 3000 RS (Thermo Fisher). Chromatographic separation was performed on a ZIC-pHILIC column (5 µm, polymeric, 150 × 4.6 mm, SeQuant®, Merck). The mobile phase (A) was 20 mM ammonium carbonate and (B) acetonitrile. The gradient program started at 80% B and was reduced to 50% B over 15 min, then reduced from 50% B to 5% B over 3 min, followed by wash with 5% B for another 3 min, and finally 8 min re-equilibration with 80% B. The flow rate was 0.3 mL/min and column compartment temperature was 40ºC. The total run time was 32 min with an injection sample volume of 10 µL.
Methods Filename:Metabolomics_pHILIC_Parkville_v1.meth
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:SeQuant ZIC-pHILIC (150 x 4.2mm,5um)
Column Pressure:60 bar at starting conditions
Column Temperature:25 C
Flow Gradient:0 min - 80%B, 15 min - 50%B, 18 min - 5%B, 21 min - 5%B, 24 min - 80%B, 32 min - 80%B
Flow Rate:0.3 ml/min
Injection Temperature:4 C
Internal Standard:CAPS, CHAPS, PIPES
Sample Injection:10 uL
Solvent A:100% water; 20 mM ammonium formate
Solvent B:100% acetonitrile
Analytical Time:32 min
Capillary Voltage:3.5 kV
Oven Temperature:25 C
Washing Buffer:syringe wash 50% IPA
Weak Wash Solvent Name:10% MeOH
Strong Wash Solvent Name:10% MeOH
Sample Loop Size:25 uL
Sample Syringe Size:25 uL
Chromatography Type:HILIC

MS:

MS ID:MS003132
Analysis ID:AN003365
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometer operated in full scan mode with positive and negative polarity switching at 35000 resolution at 200 m/z with detection range of 85 to 1, 275 m/z in full scan mode. Electro-spray ionization source (HESI) was set to 3.5 kV voltage for positive mode and 4.0 kV for negative mode, sheath gas was set to 50 and aux gas to 20 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C.
Ion Mode:POSITIVE
Capillary Temperature:300 C
Capillary Voltage:4 kV
Collision Energy:NA
Collision Gas:NA
Ion Source Temperature:120 C
Mass Accuracy:1-2 ppm
Automatic Gain Control:1e6
Dataformat:profile
Acquisition Parameters File:Metabolomics_pHILIC_Parkville_v1.pdf
Analysis Protocol File:PQMS3-MBPF-WIN-0501_analysis.pdf
  
MS ID:MS003133
Analysis ID:AN003366
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometer operated in full scan mode with positive and negative polarity switching at 35000 resolution at 200 m/z with detection range of 85 to 1, 275 m/z in full scan mode. Electro-spray ionization source (HESI) was set to 3.5 kV voltage for positive mode and 4.0 kV for negative mode, sheath gas was set to 50 and aux gas to 20 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C.
Ion Mode:NEGATIVE
Capillary Temperature:300 C
Capillary Voltage:3.5 kV
Collision Energy:NA
Collision Gas:NA
Ion Source Temperature:120 C
Mass Accuracy:1-2 ppm
Automatic Gain Control:1e6
Dataformat:profile
Acquisition Parameters File:Metabolomics_pHILIC_Parkville_v1.pdf
Analysis Protocol File:PQMS3-MBPF-WIN-0501_analysis.pdf
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