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MB Sample ID: SA045320
| Local Sample ID: | Tgon_PEPCKKO_minusglucose_13Cglutamine_4 |
| Subject ID: | SU000843 |
| Subject Type: | Cells (protozoan parasite) |
| Subject Species: | Toxoplasma gondii |
| Taxonomy ID: | 5811 |
| Genotype Strain: | RH (Type I strain) |
| Cell Biosource Or Supplier: | ATCC |
| Subject Comments: | Asexual stage (Tachyzoite) |
| Cell Passage Number: | In continuous culture |
| Cell Counts: | 10^8 cells per sample |
| Species Group: | Microorganism |
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Sample Preparation:
| Sampleprep ID: | SP000850 |
| Sampleprep Summary: | Metabolic labeling, metabolite extraction, and LC-MS profiling – DMEM minus glucose media supplemented with +613C glucose (5.5 mM) and MEM minus glutamine media supplemented with +513C-+213N glutamine (4 mM) were used as culture mediums for metabolic labeling studies using RH wt, RH ∆ku80, RH ∆hk, and RH ∆pepck1 parasites. Freshly isolated extracellular (host cell free) tachyzoites stage parasites were washed once in complete medium and resuspended in either 13C labeled glucose or glutamine (Cambridge Isotope Laboratories, Inc.) containing medium at a density of 108 parasites per milliliter. Labeling was allowed to proceed over a time period of 5, 10, 15, 30, 60 and 120 minutes in 1 ml culture suspension. At each time point, metabolites were extracted from replicate parasite samples using a modified version of a previously reported protocol. Briefly, host cell free parasites were collected by centrifugation at 3000 rpm for 5 minutes in cold and then immediately resuspended in 200 µls of ice cold 80% acetonitrile (Chem-Impex; JT Bakers) in water and incubated in ice for 15 minutes with intermittent vortexing. The supernatant was then collected after centrifuging at 13,000 rpm for 5 minutes and the pellet was further extracted twice with 100 µls of the same solvent using ultrasound in a sonicating iced water bath for 15 minutes. All the extracts were pooled (total 400 µls) and stored in -80°C until further processing for liquid chromatography coupled mass spectrometry (LC-MS) analysis. |
| Extraction Method: | Hydrophilic organic extract |
| Extract Storage: | -80 deg. Cel. until LC-MS analysis is done |
| Sample Resuspension: | The acetonitrile:water extracts from parasites were dried under nitrogen flow and resuspended in 200 µls of water:methanol (97:3) containing 10 mM tributylamine and 15 mM acetic acid. |
| Sample Derivatization: | n/a |
| Sample Spiking: | PIPES @ 100ng/ml final concentration |
