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MB Sample ID: SA316314
Local Sample ID: | 1_UI6 |
Subject ID: | SU003024 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Age Or Age Range: | Seven- to eight-week-old female BALB/c mice |
Gender: | Female |
Animal Animal Supplier: | Jackson Laboratories |
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Subject:
Subject ID: | SU003024 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Age Or Age Range: | Seven- to eight-week-old female BALB/c mice |
Gender: | Female |
Animal Animal Supplier: | Jackson Laboratories |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
1_UI6 | SA316314 | FL037609 | control | Factor |
Collection:
Collection ID: | CO003017 |
Collection Summary: | Animals were divided into three different groups: healthy controls (Mtb-, n=6), infected mice four weeks post-infection (Mtb+4w, n=6) and infected mice twelve weeks post-infection (Mtb+12w, n=5). Seven- to eight-week-old female BALB/c mice were purchased from Jackson Laboratories. Mice were housed in a pathogen-free facility with ad libitum access to water and food. All the animal studies were performed following the guidelines approved by IACUC at the University of Alabama at Birmingham, USA. Mice were aerosol infected with Mtb H37Rv using the aerosol inhalation exposure system (Glas-Col, USA) to deliver ~120-250 CFU/mouse lung. The infection dose was estimated by enumerating the lung CFU at 24 hours post-infection. Mice were sacrificed using anaesthesia with isoflurane followed by gentle cervical dislocation. Mice organs were aseptically harvested and homogenized in 2 ml of 1x PBS, pH 7.4. Serial dilutions of homogenates were prepared in 1x PBS and plated on 7H11 agar plates supplemented with 10% ADS (Albumin, Dextrose, and NaCl), Carbenicillin (25 mg/L) and Cycloheximide (25 mg/L). Plates were incubated at 37 °C for ~21 days before counting colonies. Finally, lung samples were transferred to an N2(l)-containing recipient to freeze the tissues and stored at -80 °C to avoid postmortem metabolic processes. |
Sample Type: | Lung |
Treatment:
Treatment ID: | TR003033 |
Treatment Summary: | Animals were divided into three different groups: healthy controls (Mtb-, n=6), infected mice four weeks post-infection (Mtb+4w, n=6) and infected mice twelve weeks post-infection (Mtb+12w, n=5). Seven- to eight-week-old female BALB/c mice were purchased from Jackson Laboratories. Mice were housed in a pathogen-free facility with ad libitum access to water and food. All the animal studies were performed following the guidelines approved by IACUC at the University of Alabama at Birmingham, USA. Mice were aerosol infected with Mtb H37Rv using the aerosol inhalation exposure system (Glas-Col, USA) to deliver ~120-250 CFU/mouse lung. The infection dose was estimated by enumerating the lung CFU at 24 hours post-infection. Mice were sacrificed using anaesthesia with isoflurane followed by gentle cervical dislocation. Mice organs were aseptically harvested and homogenized in 2 ml of 1x PBS, pH 7.4. Serial dilutions of homogenates were prepared in 1x PBS and plated on 7H11 agar plates supplemented with 10% ADS (Albumin, Dextrose, and NaCl), Carbenicillin (25 mg/L) and Cycloheximide (25 mg/L). Plates were incubated at 37 °C for ~21 days before counting colonies. Finally, lung samples were transferred to an N2(l)-containing recipient to freeze the tissues and stored at -80 °C to avoid postmortem metabolic processes. |
Sample Preparation:
Sampleprep ID: | SP003030 |
Sampleprep Summary: | The sample preparation and lipid extraction were performed at the Centers for AIDS Research and Free Radical Biology, University of Alabama at Birmingham (Birmingham, AL, United States), following a protocol initially described and optimized at CEMBIO (Madrid, Spain). Approximately 75 mg of lung tissue was mixed with a cold (–20°C) mixture of MeOH:H2O (1:1, v/v) added in a ratio of 1 mg tissue:10 µL of extraction solvent. Next, the tissue samples were homogenized using the Dounce homogenizer. After the homogenization, 200 µL of homogenate was mixed with 640 µL of MeOH and 160 µL of Methyl-Tert-Butyl ether (MTBE) to extract hydrophobic compounds. Samples were then vortex-mixed for 1 hour at room temperature (RT) and centrifuged at 4000 g for 20 min at 20°C. The samples were then passed through spin X columns (0.22 µm filter), and 200 µL of the filtered sample was dried at RT in the vacuum concentrator. From here, the samples were sent to CEMBIO for the UHPLC-MS analysis. Prior to the analysis, dried samples were re-suspended with 200 µL of MeOH/MTBE/H2O (7.4:1.6:1, v/v/v), which contained the corresponding ISs (C17 – sphingosine at 1 ppm for positive ion mode, and d31–palmitic acid at 3 ppm for negative ion mode). Samples were then centrifuged (16,100xg, 5 min, 15°C) before transferring them into sample vials with glass inserts for LC-MS analysis. |
Combined analysis:
Analysis ID | AN004780 | AN004781 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Reversed phase | Reversed phase |
Chromatography system | Agilent 1290 Infinity II | Agilent 1290 Infinity II |
Column | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) |
MS Type | ESI | ESI |
MS instrument type | QTOF | QTOF |
MS instrument name | Agilent 6545 QTOF | Agilent 6545 QTOF |
Ion Mode | POSITIVE | NEGATIVE |
Units | AREA | AREA |
Chromatography:
Chromatography ID: | CH003612 |
Instrument Name: | Agilent 1290 Infinity II |
Column Name: | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) |
Column Temperature: | 50 °C |
Flow Gradient: | Started at 70% of B at 0 –1 min, 86% at 3.5 –10 min, and 100% B at 11–17 min |
Flow Rate: | 0.6 mL/min |
Solvent A: | 90% water/10% methanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride |
Solvent B: | 20% acetonitrile/30% methanol/50% isopropanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS004526 |
Analysis ID: | AN004780 |
Instrument Name: | Agilent 6545 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | 150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was infused throughout the whole analysis: purine (C5H4N4) at m/z 121.0509 and HP-0921 (C18H18O6N3P3F24) at m/z 922.0098. These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction. |
Ion Mode: | POSITIVE |
MS ID: | MS004527 |
Analysis ID: | AN004781 |
Instrument Name: | Agilent 6545 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | 150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was infused throughout the whole analysis: purine (C5H4N4) at m/z 119.0363 and HP-0921 (C18H18O6N3P3F24) at m/z 980.0163 (HP-0921 + acetate). These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction. |
Ion Mode: | NEGATIVE |