13C Kinetic Labeling and Extraction of Metabolites from Adherent Mammalian Cells
13C 动力标记和提取附着哺乳动物细胞的代谢产物
发布: 2015年04月20日第5卷第8期 DOI: 10.21769/BioProtoc.1447 浏览次数: 11472
评审: Fanglian HeValentine V TrotterKanika Gera
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参见作者原研究论文
The authors used this protocol in:
Jul 2014
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Abstract
Fluctuations in metabolite levels in mammalian cells are the most direct form of readout of the cellular metabolic state. The current protocol describes a method for pulse labeling and subsequent isolation of metabolites from adherent mammalian cells. The isolated metabolites can be identified and quantified by mass-spectrometry, allowing for estimation of the rates of synthesis and removal of metabolites from the system being analyzed.
Materials and Reagents
- THP1 cells (from ATCC, catalog number: TIB 202 )
- Methanol (LC-MS Ultra CHROMASOLV) (Sigma-Aldrich, catalog number: 14262 )
- Water (LC-MS Ultra CHROMASOLV) (Sigma-Aldrich, catalog number: 14263 )
- Acetonitrile (LC-MS Ultra CHROMASOLV) (Sigma-Aldrich, catalog number: 14261 )
- Hexane (Sigma-Aldrich, catalog number: 296090 )
- HCl (Sigma-Aldrich, catalog number: 258148 )
- Trypan blue (Gibco, catalog number: 15250-061 )
- Phorbol myristate acetate (PMA) (Sigma-Aldrich, catalog number: P8139 )
- Pyridine (Sigma-Aldrich, catalog number: 270970 )
- Sulfur trioxide pyridine (Sigma-Aldrich, catalog number: S7556 )
- Barium acetate (Sigma-Aldrich, catalog number: 243671 )
- D-Glucose (13C6 99%) (Euriso-Top, catalog number: CLM-481 )
- RPMI culture media 1640 (Gibco, catalog number: 31800-014 )
- Glucose free RPMI media-1640 (Sigma-Aldrich, catalog number: R1383 )
- Dialyzed FBS (Hyclone, catalog number: SH30071.03 )
- Cholate buffer (see Recipes)
- Quenching mix (see Recipes)
Equipment
- Bright-line hemacytometer (Sigma-Aldrich, catalog number: Z359629 )
- Cell scraper
- Culture dish
6 well plate (Nunc, catalog number: 140685 )
Flask (T-175, catalog number: 178983 ) - Liquid nitrogen cylinder
- Nitrogen gas cylinder
- Water bath
- Refrigerators (-20, -80, 4 °C)
- Microcentrifuge (Eppendrof, model: 5418R )
- Mono spin C18 plugs (GL Sciences, catalog number: 5010-21700 )
- HPLC (Agilent, model: 1260 infinity ; Pump: Binary Pump VL, model: G1312C )
- HPLC column
Amino column (Polaris 5 NH2 150 * 2.0 mm)
C18 column [ZORBAX Eclipse Plus C18 (Narrow Bore 2.1 * 150 mm 5 μ)]
Cyano column (Phenomenax Luna 150 * 2.0 mm 3 μ) - Mass spectrometer (ABSciex, hybrid 4000 QTrap)
- Amber glass vials (Supelco, catalog number: 29117 )
- Glass vials (Borosil, catalog number: 9910 )
- Glass pasture pipette
- Sonicator (Branson, model: 1210 )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Mehrotra, P., Saquib, N. and Rao, K. V. S. (2015). 13C Kinetic Labeling and Extraction of Metabolites from Adherent Mammalian Cells. Bio-protocol 5(8): e1447. DOI: 10.21769/BioProtoc.1447.
- Mehrotra, P., Jamwal, S. V., Saquib, N., Sinha, N., Siddiqui, Z., Manivel, V., Chatterjee, S. and Rao, K. V. (2014). Pathogenicity of Mycobacterium tuberculosis is expressed by regulating metabolic thresholds of the host macrophage. PLoS Pathog 10(7): e1004265.
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分类
生物化学 > 脂质 > 脂质分离
细胞生物学 > 细胞新陈代谢 > 脂质
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