Return to study ST003047 main page
MB Sample ID: SA330839
| Local Sample ID: | WT_C |
| Subject ID: | SU003162 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003162 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| WT_C | SA330839 | FL039173 | Wild-type | Genotype |
Collection:
| Collection ID: | CO003155 |
| Collection Summary: | Tfamfl/fl mice (Larsson et al., 1998) were provided by Larsson NG (Max Plack Institute for Biology of Ageing, Cologne, Germany) and were crossed at the Centro de Biologia Molecular Severo Ochoa (CBMSO) animal facility with MB1Cre mice (Hobeika et al., 2006). MB1Cre mice were kindly provided by Alarcón B (CBMSO, Madrid, Spain). Mouse colonies were bred at the CBMSO under specific pathogen-free conditions and on C57BL/6 background. Mice were group-housed, have not been used in previous procedures and were fed with standard chow. Littermates were randomly assigned to experimental groups. Male and female between the ages of 8-16 weeks were used for the experiment. Splenic naive B lymphocytes were purified using negative B cell cell isolation kits (Miltenyi Biotec). After 24 hours stimulation, live cells were sorted, pelleted and frozen with liquid N2. |
| Collection Protocol Filename: | Protocol_BL.pdf |
| Sample Type: | B-cells |
Treatment:
| Treatment ID: | TR003171 |
| Treatment Summary: | Mice were group-housed and fed with standard chow. B lympchocytes from wild-type and knock-out mice were isolated and cultured for 24 hours in RPMI 1640 supplemented with 10% FCS, 1mM Glutamine, 0,01% sodium pyruvate, 20mM 2-mercaptoethanol, penicillin and streptomycin. Cells were cultured adding interleukine-4 and interleukine-5 at 10 ng/Ml, anti-IgM 1 ug/mL and anti-CD40 0,3 ug/mL. |
Sample Preparation:
| Sampleprep ID: | SP003168 |
| Sampleprep Summary: | From wild-type samples WT_A, WT_B and WT_C 5.6 x 106, 6.4 x 106 and 4.87 x 106 B lymphocytes were harvested respectively, and for knock-out samples KO_D, KO_E and KO_F 1.8 x 106, 2.1 x 106 and 1.8 x 106 B lymphocytes were harvested respectively. Cell homogenates were prepared by adding 1:1 v/v water: methanol and sonicated with a dr.hielscher UP200S (dr.hielscher, Berlin, Germany), set up to 80% intensity, 0.5 s/pulse, 16 pulses. For GC-MS, 100 µL of each homogenate was mixed with 300 µL of cold methanol containing 25 ppm D-palmitic acid (internal standard), vortex mixed for 60 min and centrifuged at 4000 g for 20 min. 250 µL of the supernatant were transferred to a vial with a glass insert and evaporated to dryness in a Gyrozen HyperVac VC2124 (Gimpo, Korea) coupled to a LVS 110Z (Gardner Denver Thomas GmbH Welch Vacuum, Fürstenfeldbruck, Germany). The samples were then submitted to methoximation (with O-methoxyamine) for 16 h and silylation for 1 h at 70ºC with N,O-Bis(trime5j,thylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TCMS), and finally resuspended in 100 µL heptane. |
| Sampleprep Protocol Filename: | Protocol_BL.pdf |
Combined analysis:
| Analysis ID | AN004998 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 8890 |
| Column | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| MS Type | EI |
| MS instrument type | GC-Q-MS |
| MS instrument name | Agilent 5977B |
| Ion Mode | POSITIVE |
| Units | relative abundance |
Chromatography:
| Chromatography ID: | CH003776 |
| Chromatography Summary: | For GC-MS, the samples were injected onto a DB5-MS column (30 m x 0.250 mm, 0.25 µm) with a 10 m pre-column (Agilent Technologies, Santa Clara CA, USA). Helium was used as mobile phase at constant flow rate (1 mL/min) in a GC-Q-MS (8890 GC coupled to 5977B MS) system (Agilent Technologies) with an Electron Ionization (EI) source at 70 eV. |
| Methods Filename: | Protocol_BL.pdf |
| Instrument Name: | Agilent 8890 |
| Column Name: | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| Column Temperature: | -60 °C-325/350 °C |
| Flow Gradient: | NA |
| Flow Rate: | 1 mL/min |
| Solvent A: | NA |
| Solvent B: | NA |
| Chromatography Type: | GC |
MS:
| MS ID: | MS004738 |
| Analysis ID: | AN004998 |
| Instrument Name: | Agilent 5977B |
| Instrument Type: | GC-Q-MS |
| MS Type: | EI |
| MS Comments: | Cell homogenates were prepared by adding 1:1 v/v water: methanol and sonicated with a dr.hielscher UP200S (dr.hielscher, Berlin, Germany), set up to 80% intensity, 0.5 s/pulse, 16 pulses. For GC-MS, 100 μL of each homogenate was mixed with 300 μL of cold methanol containing 25 ppm D-palmitic acid (internal standard), vortex mixed for 60 min and centrifuged at 4000 g for 20 min. 250 μL of the supernatant were transferred to a vial with a glass insert and evaporated to dryness in a Gyrozen HyperVac VC2124 (Gimpo, Korea) coupled to a LVS 110Z (Gardner Denver Thomas GmbH Welch Vacuum, Fürstenfeldbruck, Germany). The samples were then submitted to methoximation (with O-methoxyamine) for 16 h and silylation for 1 h at 70ºC with N,O-Bis(trime5j,thylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TCMS), and finally resuspended in 100 μL heptane. The samples were injected onto a DB5-MS column (30 m x 0.250 mm, 0.25 μm) with a 10 m pre-column (Agilent Technologies, Santa Clara CA, USA). Helium was used as mobile phase at constant flow rate (1 mL/min) in a GC-Q-MS 5975 system (Agilent Technologies) with an Electron Ionization (EI) source at 70 eV. |
| Ion Mode: | POSITIVE |
