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MB Sample ID: SA237015
| Local Sample ID: | Ucp2PomcKO_2_RP_Pos |
| Subject ID: | SU002450 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002450 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Ucp2PomcKO_2_RP_Pos | SA237015 | FL029344 | UCP2 POMC-knockout | Genotype |
Collection:
| Collection ID: | CO002443 |
| Collection Summary: | Hypothalamic primary neuronal cell culture Eight to ten neonatal (0 days old) pubs from either control or Ucp2PomcKO mice derived from homozygous Cre-positive parents were used for hypothalamic primary neuronal cell culture. In brief, we carefully removed the brain's hypothalamus and placed it onto a small culture dish containing a small volume of Hibernate-A Medium (Cat# A1247501, Thermo Fisher Scientific). After digestion, the tissues dissociated to single cells with 6 mL of Hibernate-A Medium containing 2.5 % of Trypsin-EDTA for 15 minutes at 37℃. Suspended cells were filtered (40 μm) and centrifuged for 5 min at 1000 rpm. The pellet was re-suspended and plated on XF96 cell culture microplates (Cat# 101085-004, Agilent Technologies) coated with poly-D-lysine (Cat# P6407, Sigma-Aldrich) at a density of 0.2 x104 cells per well. Cells were cultured in Neurobasal-A medium (Cat# 10888022, Thermo Fisher Scientific) supplemented with 1 % penicillin-streptomycin, 2 % B-27 Supplement (Cat# 17504044, Thermo Fisher Scientific), and GlutaMAX-I (Cat# 35050061, Thermo Fisher Scientific), CultureOne supplement (Cat# A3320201, Thermo Fisher Scientific). For control culture, we used either Ucp2fl/fl; Pomc-CreERT2; tdTomato mice which neuronal cultures were treated with vehicle (0.03 % ethanol diluted in medium) or Ucp2+/+; Pomc-CreERT2; tdTomato mice which neuronal cultures were treated with 2 μM 4-hydroxytamoxifen (Cat# H7904, Sigma-Aldrich). After 10 days in culture, primary neuronal cells isolated from control and Ucp2PomcKO mice were treated with 2 μM 4-hydroxytamoxifen for expression of a CreER recombinase. Primary neuronal cells were used for the measurement of mitochondria oxidation two days later. |
| Sample Type: | Neurons |
Treatment:
| Treatment ID: | TR002462 |
| Treatment Summary: | We used the inducible Cre/loxP technology to generate mice in which UCP2 was selectively ablated in POMC neurons (Ucp2PomcKO mice). First, mice expressing a tamoxifen-inducible Cre recombinase (CreERT2) in cells expressing POMC (Pomc-CreERT2) were crossed with Rosa26-lox-stop-lox-tdTomato (Ai14; cre-recombinase-dependent expression) mice (Ai14 reporter mice; stock #007914; The Jackson Laboratory) to label POMC-expressing cells. Pomc-CreERT2; Rosa26-lox-stop-lox-tdTomato (Pomc-CreERT2; tdTomato) mice have POMC-expressing cells with the expression of tdTomato by tamoxifen administration. No observation of POMC-tdTomato expression was found in the absence of tamoxifen administration, indicating that recombination was strictly dependent upon tamoxifen-induced Cre recombinase activation. The mice with Pomc-CreERT2; tdTomato were then crossed with mice harboring conditional alleles Ucp2 floxed (Ucp2fl/fl; B6;129S-Ucp2tm2.1Lowl/J, Stock# 022394; The Jackson Laboratory) to generated mice with inducible deletion of Ucp2 specifically in POMC neurons (Ucp2PomcKO mice). All animal experiments were conducted with the mice expressing POMC-CreERT2. As control groups, Ucp2fl/fl; Pomc-CreERT2 mice were injected with tamoxifen, and Ucp2fl/fl; Pomc-CreERT2; tdTomato were mice injected with corn oil (vehicle), and Ucp2fl/fl; Pomc-CreERT2; tdTomato mice were injected intraperitoneally (IP) with tamoxifen (0.1 mg/g BW for 5 consecutive days) starting at 5 weeks of age to induce mature-onset deletion of Ucp2 in POMC neurons of Ucp2PomcKO mice. However, because there were no differences between these two control groups, most of the experiments were performed using Ucp2+/+; Pomc-CreERT2; tdTomato mice injected with tamoxifen (to label POMC neurons with tdTomato expression) as a control group, unless otherwise stated. Body composition was measured in vivo by MRI (EchoMRI; Echo Medical Systems, Houston, TX) monthly at 10:00 AM. We performed transcriptomic profiling by using a ribosomal tagging strategy to analyze POMC neuron-specific mRNA expression in vivo. We crossed Pomc-CreERT2 mice30 with Rpl22 floxed (RiboTag, Stock# 029977, The Jackson Laboratories) mice to generate Pomc-CreERT2; RiboTag mice, expressing a hemagglutinin A (HA)-tagged ribosomal protein in the POMC neurons upon tamoxifen injection. |
Sample Preparation:
| Sampleprep ID: | SP002456 |
| Sampleprep Summary: | Cells cultured in 6-well plates were quickly washed once with ice cold 5 mM HEPES, then immediately quenched in 150ul/well ice cold quench buffer (20% methanol, 3 mM sodium fluoride, 0.1% formic acid, 1mM phenylalanine and 100uM EDTA). Each well was harvested using a cell lifter and immediately transferred to a pre-chilled 96-well plate on dry ice. Once completely frozen, cell lysates were lyophilized and stored in the -80oC freezer until the sample run. Lyophilized samples were prepared by resuspending in 50 µL water with D4-taurine (50 µM) as an internal standard and 5 µL of the supernatant was injected for each analysis mode using high performance liquid chromatography (HPLC) into the 6600 Triple TOF LC-MS/MS mass spectrometer (Sciex). |
Combined analysis:
| Analysis ID | AN003855 | AN003856 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Unspecified |
| Chromatography system | SCIEX ExionLC | SCIEX ExionLC |
| Column | Phenomenex Kinetex F5 Core-shell LC (100 x 2.1 mm,2.6um) | Thermo Scientific Hypercarb (100 x 4.6 mm,3um) |
| MS Type | ESI | ESI |
| MS instrument type | Triple TOF | Triple TOF |
| MS instrument name | ABI Sciex 6600 TripleTOF | ABI Sciex 6600 TripleTOF |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH002853 |
| Chromatography Summary: | Reverse phase column with positive MS mode |
| Instrument Name: | SCIEX ExionLC |
| Column Name: | Phenomenex Kinetex F5 Core-shell LC (100 x 2.1 mm,2.6um) |
| Column Temperature: | 30 |
| Flow Rate: | 0.3ml/min |
| Solvent A: | 95% water/5% acetonitrile; 0.1% formic acid |
| Solvent B: | 95% acetonitrile/5% water; 0.1% formic acid |
| Analytical Time: | 20min |
| Washing Buffer: | 50%Methanol, 50% water |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002854 |
| Chromatography Summary: | Hypercarb column with negative MS mode |
| Instrument Name: | SCIEX ExionLC |
| Column Name: | Thermo Scientific Hypercarb (100 x 4.6 mm,3um) |
| Column Temperature: | 50 |
| Flow Rate: | 1ml/min |
| Solvent A: | 100% water; 15 mM ammonium formate; 0.03% acetyl acetone; 0.1% formic acid |
| Solvent B: | 60% acetonitrile/35% isopropanol; 0.1% formic acid; 15 mM ammonium formate |
| Analytical Time: | 18min |
| Washing Buffer: | 50%Methanol, 50% water |
| Chromatography Type: | Unspecified |
MS:
| MS ID: | MS003596 |
| Analysis ID: | AN003855 |
| Instrument Name: | ABI Sciex 6600 TripleTOF |
| Instrument Type: | Triple TOF |
| MS Type: | ESI |
| MS Comments: | Data were collected using an information-dependent analysis (IDA) workflow consisting of a TOF MS scan (200 msec) and a high-resolution IDA experiment (70 msec each) monitoring 10 candidate ions per cycle. Former target ions were excluded after 2 occurrences for 5 seconds and dynamic background subtraction was employed. The mass range for both TOF MS and IDA MS/MS scans was 60-1000. Ion spray voltage = 5000 V; ion source gas 1 (GS1) = 50 psi, ion source gas 2 (GS2) = 50 psi, curtain gas (CUR) = 30 psi, temperature (TEM) = 400 oC. declustering potential (DP) = 35 V, collision energy (CE) = 30 V, collision energy spread (CES) = 20 V. El-MAVEN software (Elucidata.io) was used for peak picking and curation from house built targeted and untargeted libraries. Targeted libraries were developed using a commercial standard kit of ~600 metabolites (IROA Technologies) in each mode, yielding reference data (molecular ions and retention times) with wide coverage of the endogenous metabolome. Untargeted libraries contained ~2,700 metabolites drawn from the KEGG database where the top 5 candidates with the highest intensity throughout a sample run for each metabolite were automatically curated. |
| Ion Mode: | POSITIVE |
| MS ID: | MS003597 |
| Analysis ID: | AN003856 |
| Instrument Name: | ABI Sciex 6600 TripleTOF |
| Instrument Type: | Triple TOF |
| MS Type: | ESI |
| MS Comments: | Data were collected using an information-dependent analysis (IDA) workflow consisting of a TOF MS scan (200 msec) and a high-resolution IDA experiment (70 msec each) monitoring 10 candidate ions per cycle. Former target ions were excluded after 2 occurrences for 5 seconds and dynamic background subtraction was employed. The mass range for both TOF MS and IDA MS/MS scans was 70-1000. Ion spray voltage = -4500V, ion source gas 1 (GS1) = 50 psi, ion source gas 2 (GS2) = 50 psi, curtain gas (CUR) = 30 psi, temperature (TEM) = 500 oC.declustering potential (DP) = 35 V, collision energy (CE) = 30 V, collision energy spread (CES) = 20 V. El-MAVEN software (Elucidata.io) was used for peak picking and curation from house built targeted and untargeted libraries. Targeted libraries were developed using a commercial standard kit of ~600 metabolites (IROA Technologies) in each mode, yielding reference data (molecular ions and retention times) with wide coverage of the endogenous metabolome. Untargeted libraries contained ~2,700 metabolites drawn from the KEGG database where the top 5 candidates with the highest intensity throughout a sample run for each metabolite were automatically curated. |
| Ion Mode: | NEGATIVE |
