13C targeted metabolomics
Ontology highlight
ABSTRACT:
ORGANISM(S): Human Homo Sapiens
TISSUE(S): T-cells
SUBMITTER: 
Maureen Kachman
PROVIDER: ST000147 | MetabolomicsWorkbench | Fri Mar 13 00:00:00 GMT 2015
REPOSITORIES: MetabolomicsWorkbench
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| mwtab | Other |
Items per page: 5 1 - 1 of 1 |
Similar Datasets
Project description:This study provides a comprehensive evaluation of changes in gene expression during treatment with Genistein in vitro. Ishikawa cells were maintained and grown (to confluency or at a desired cell density between 500,000 - 1 million cells/mL) in DMEM/F-12 Medium (supplemented with 10% Fetal Bovine Serum + 1X penicillin/streptomycin) in xenoestrogen-free Corning plasticware. Cells were gently washed in warm PBS and transferred to phenol red-free DMEM/F-12 Medium (supplemented with 10% Charcoal-Stripped Fetal Bovine Serum + 1X penicillin/streptomycin) in Corning Cell Culture Cluster wells overnight and then challenged with 10pM (very low, vL), 1 nM (low, L), 10nM (high, H), and 1uM (very high, vH) levels of Genistein. Prior to collection, cells were washed in warm PBS, resuspended & briefly incubated in TRI-Reagent, & finally collected (in quintuplicate replicates) at time each specific time point: 8 hours, 24 hours, and 48 hours. Following RNA isolation, the best RNA yields for each quadruplicate set was selected for target preparation and GeneChip processing.
2016-07-22 | GSE71717 | GEO
Project description:This study provides a comprehensive evaluation of changes in gene expression during treatment with Bisphenol A in vitro. Ishikawa cells were maintained and grown (to confluency or at a desired cell density between 500,000 - 1 million cells/mL) in DMEM/F-12 Medium (supplemented with 10% Fetal Bovine Serum + 1X penicillin/streptomycin) in xenoestrogen-free Corning plasticware. Cells were gently washed in warm PBS and transferred to phenol red-free DMEM/F-12 Medium (supplemented with 10% Charcoal-Stripped Fetal Bovine Serum + 1X penicillin/streptomycin) in Corning Cell Culture Cluster wells overnight and then challenged with 1pM (very low, vL), 100pM (low, L), 10nM (high, H), and 1uM (very high, vH) levels of BPA. Prior to collection, cells were washed in warm PBS, resuspended & briefly incubated in TRI-Reagent, & finally collected (in quintuplicate replicates) at each specific time point: 8 hours, 24 hours, and 48 hours. Following RNA isolation, the best RNA yields for each quadruplicate set was selected for target preparation and GeneChip processing. Keywords: Dose Response/Time Course
2009-08-13 | GSE17624 | GEO
Project description:Microarray expression arrays on THP-1 acute myeloid leukemia cell line samples, contrasting expression profiles of freshly harvested cell pellets, cells replated into fresh media for 24 hours and cells directly pelleted from liquid nitrogen stocks.
2012-12-25 | GSE30819 | GEO
Project description:ICR mice, age 6-8 weeks.(Harlan, Indianapolis, IN, USA). Housed individually with controlled temperature, humidity, and light-dark cycle (0600h – 1800h). Pregnant mice were housed individually with ad libitum access to standard rodent chow (T.2018, Harlan Teklad, Indianapolis, IN, USA). On day 12.5 of pregnancy, dams were randomly assigned to either a control or undernutrition group. In the undernutrition group, food was restricted to 50% that of gestational day -matched controls for the remainder of pregnancy. After delivery, mothers were given ad libitum access to chow and 24 hours after birth, litters were equalized to eight. We studied mice from 2 groups: in utero undernourished offpring (U) and control offspring (C). Primary muscle cells were isolated from the quadriceps of U and C at 3 weeks old. Isolated primary muscle cells were grown on Matrigel coated flasks in low glucose (5.5 mmol/L) Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20% FBS, 1X antibiotic-antimycotic, 2.5μg/ml gentamycin and 2.5ng/ml basic fibroblast growth factor. When cells reached approximately 90% confluency, they were differentiated in low glucose DMEM supplemented with 2% FBS, 1X antibiotic-antimycotic, and 2.5μg/ml gentamycin. Other cells were cultured overnight in substrate limited medium (glucose and glutamine free DMEM supplemented with 0.5mM glucose, 1mM glutamine, 0.5mM carnitine and 1% (v/v) FBS; pH 7.4).
2019-09-12 | GSE103689 | GEO
Project description:This study provides a comprehensive evaluation of changes in gene expression during treatment with Genistein in vitro. Ishikawa cells were maintained and grown (to confluency or at a desired cell density between 500,000 - 1 million cells/mL) in DMEM/F-12 Medium (supplemented with 10% Fetal Bovine Serum + 1X penicillin/streptomycin) in xenoestrogen-free Corning plasticware. Cells were gently washed in warm PBS and transferred to phenol red-free DMEM/F-12 Medium (supplemented with 10% Charcoal-Stripped Fetal Bovine Serum + 1X penicillin/streptomycin) in Corning Cell Culture Cluster wells overnight and then challenged with 10pM (very low, vL), 1 nM (low, L), 10nM (high, H), and 1uM (very high, vH) levels of Genistein. Prior to collection, cells were washed in warm PBS, resuspended & briefly incubated in TRI-Reagent, & finally collected (in quintuplicate replicates) at time each specific time point: 8 hours, 24 hours, and 48 hours. Following RNA isolation, the best RNA yields for each quadruplicate set was selected for target preparation and GeneChip processing. 60 total samples: 5 Doses [Vehicle Control, Very Low (1 pM), Low (100 pM), High (1 nM), Very High (1 uM)]; 3 Timepoints [8 hour, 24 hour, 48 hour]; 4 Replicates each
2016-07-22 | E-GEOD-71717 | biostudies-arrayexpress
Project description:This study provides a comprehensive evaluation of changes in gene expression during treatment with Bisphenol A in vitro. Ishikawa cells were maintained and grown (to confluency or at a desired cell density between 500,000 - 1 million cells/mL) in DMEM/F-12 Medium (supplemented with 10% Fetal Bovine Serum + 1X penicillin/streptomycin) in xenoestrogen-free Corning plasticware. Cells were gently washed in warm PBS and transferred to phenol red-free DMEM/F-12 Medium (supplemented with 10% Charcoal-Stripped Fetal Bovine Serum + 1X penicillin/streptomycin) in Corning Cell Culture Cluster wells overnight and then challenged with 1pM (very low, vL), 100pM (low, L), 10nM (high, H), and 1uM (very high, vH) levels of BPA. Prior to collection, cells were washed in warm PBS, resuspended & briefly incubated in TRI-Reagent, & finally collected (in quintuplicate replicates) at each specific time point: 8 hours, 24 hours, and 48 hours. Following RNA isolation, the best RNA yields for each quadruplicate set was selected for target preparation and GeneChip processing. Experiment Overall Design: 60 total samples: 5 Doses [Vehicle Control, Very Low (1 pM), Low (100 pM), High (10 nM), Very High (1 uM)]; 3 Timepoints [8 hour, 24 hour, 48 hour]; 4 Replicates each.
2009-08-23 | E-GEOD-17624 | biostudies-arrayexpress
Project description:A mouse hepatoma cell line (Hepa1-6 cells, ATCC) was grown in T-25 tissue culture flasks (Corning) at 37ºC in 5% CO2 under two conditions. The first condition was the control, which contained DMEM medium (Gibco BRL) with 25 mM glucose, 4 mM glutamine, 1% penicillin-streptomycin (Sigma), and 10% CPSR-3 (Sigma). The second condition was identical to the first, however the glutamine concentration was altered periodically. Once the cells had grown to confluency, the experiment began at 9:30 PM by exchanging the medium in all flasks. The control flasks received the same medium, containing 4 mM glutamine, while the experimental flasks received identical medium with no glutamine. Twenty-four hours later, the medium was exchanged again, however this time all flasks received medium containing 4 mM glutamine. The first medium exchange was repeated at 48 hours into the experiment, while the second was repeated at 72 hours into the experiment. Hence the glutamine concentration of the experimental flasks oscillated between 0 mM and 4 mM glutamine over 96 hours, while the glutamine concentration of the control flasks remained constant at 4 mM glutamine. At regular intervals, the flasks were sampled. Total RNA was isolated, medium samples were retained, and isotopic measurements were performed from flasks fed different labelled compounds. All samples taken during the experiment were analyzed. The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples from identical time points that were to be compared on DNA microarrays were then mixed. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65ºC. Following concentration, the sample was resuspended in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), 1 uL was removed for quantitation, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.). The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene). The slides were then blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride, 239 mL 1-methyl-2-pyrrolidinone, and 10.7 mL of 1 M boric acid, pH 8.0. They were cleaned by rinsing in 250 mL of milli-Q water (X2), followed by 250 mL of 95% ethanol. They were finally dried using filtered air and stored in the dark at room temperature. Keywords = mouse, liver, hepatoma, cell culture, glutamine, carbon metabolism Keywords: time-course
2003-04-16 | GSE404 | GEO
Project description:The human acute lymphoblastic leukemia cell line (Jurkat) was used to isolate a clones based on Noxa expression. JP6 is low Noxa, JP3 is high Noxa. Furthermore, a Noxa expressing plasmid was tranfected and stable clones were selected for a Noxa high model (N5). 10E6 cells were starved of glucose (A) or glutamine (B&C) for 3 hours and then fed 13C 1,2 glucose (A), 15N alpha nitrogen glutamine (B) or 15N amide nitrogen glutamine (C) for 24 hours. Cells were washed 1X in ice cold PBS and resuspended in -20C methanol. Quenched cells were snap frozen and stored at -80. For experiment A we would like to observe the contribution of labeled glucose into the synthesis of amino acids, specifically glycine and serine. For experiment B we are most interested in the nitrogen incorporation into aspartate. SCB edits: This fluxomics study requires measuring amino acids for M, M+1, M+2, and M+3 prioritizing glycine, serine, aspartate, asparagine, ornithine, citrulline, then as many as possible using a diamond hydride column and LCMS method (see CEvans).
2015-01-21 | ST000154 | MetabolomicsWorkbench
Project description:A mouse hepatoma cell line (Hepa1-6 cells, ATCC) was grown in T-25 tissue culture flasks (Corning) at 37ºC in 5% CO2 under two conditions. The first condition was the control, which contained DMEM medium (Gibco BRL) with 25 mM glucose, 4 mM glutamine, 1% penicillin-streptomycin (Sigma), and 10% CPSR-3 (Sigma). The second condition was identical to the first, however the glutamine concentration was altered periodically. Once the cells had grown to confluency, the experiment began at 9:30 PM by exchanging the medium in all flasks. The control flasks received the same medium, containing 4 mM glutamine, while the experimental flasks received identical medium with no glutamine. Twenty-four hours later, the medium was exchanged again, however this time all flasks received medium containing 4 mM glutamine. The first medium exchange was repeated at 48 hours into the experiment, while the second was repeated at 72 hours into the experiment. Hence the glutamine concentration of the experimental flasks oscillated between 0 mM and 4 mM glutamine over 96 hours, while the glutamine concentration of the control flasks remained constant at 4 mM glutamine. At regular intervals, the flasks were sampled. Total RNA was isolated, medium samples were retained, and isotopic measurements were performed from flasks fed different labelled compounds. All samples taken during the experiment were analyzed. The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples from identical time points that were to be compared on DNA microarrays were then mixed. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65ºC. Following concentration, the sample was resuspended in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), 1 uL was removed for quantitation, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.). The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene). The slides were then blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride, 239 mL 1-methyl-2-pyrrolidinone, and 10.7 mL of 1 M boric acid, pH 8.0. They were cleaned by rinsing in 250 mL of milli-Q water (X2), followed by 250 mL of 95% ethanol. They were finally dried using filtered air and stored in the dark at room temperature. Keywords = mouse, liver, hepatoma, cell culture, glutamine, carbon metabolism
2010-06-05 | E-GEOD-404 | biostudies-arrayexpress
Project description:3x107 cells were harvested and washed in 30 ml cold 1x TDB. The pellet was resuspended in 300 µl permeabilization buffer with protease inhibitors, 3 µl of 4 mM digitonin was added and incubated for 5 minutes at RT. The cells were pelleted, resuspended in 600 µl isotonic buffer with protease inhibitors and split in two samples, containing 1x107 and 2x107 cells, respectively. The transposition reaction was performed by adding 50 µl of transposition mix to the pellet (25 µl TD (2x reaction buffer from Nextera kit), 25 µl TDE1 (Tn5 transposase from Nextera kit), 22.5 µl nuclease-free water) and incubation for 30 minutes at 37 °C. For the gDNA control, 200 ng of gDNA was treated in the same manner. The DNA samples were purified using Qiagen MinElute PCR Purification Kit and eluted in 10 µl EB (10 mM Tris-HCl, pH 8). The transposed DNA fragments were amplified using the NEBNext® High-Fidelity 2X PCR Master Mix (M0541) supplied with 2.5 µl of 25 µM barcoded primers and amplification for 13 cycles. The libraries were purified using AMPure XP beads (Beckman Coulter) following the manufacturer’s instructions. The library fragment sizes between 150 and 1000 bp were purified from a 6% polyacrylamide gel. Paired-end 76 bp sequencing was carried out using the Illumina NextSeq system with a mid-output NextSeq 500/550 kit according to the manufacturer’s instructions.
2018-08-24 | GSE118902 | GEO