The SBP Cancer Metabolism Core provides a hub within the Institute for analysis of metabolism using specialist instruments and methodologies, together with advice on planning experiments and analyzing data. All of the instruments and approaches are available on a fee-for-service basis to Cancer Center scientists, but also (as capacity permits) to outside users. Samples for most services can be shipped, but Seahorse analyzer users need to be local so they can bring live cells for analysis.
The primary services offered by the core are:
- GC/MS-based analysis of metabolites, including (1) a panel of 30-40 small polar metabolites (such as amino acids, components of glycolysis and TCA pathways); (2) fatty acids and cholesterol; (3) sugars and sugar alcohols and acids; (4) sugar phosphates; (5) short-chain fatty acids. We do both quantification versus standards and stable isotope (13C, 15N, 2H) tracing, the combination of which can give powerful insights into metabolic activity.
- Measurement of cellular respiration and glycolysis using a Seahorse XFp. This instrument determines the rate of glycolysis and respiration of adherent cells in a 6-well format. Glycolysis is observed via the acidification of the tissue culture media. Respiration is observed by measuring total oxygen consumption. With proper experimental design, the rates of basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity and non-mitochondrial oxygen consumption may be determined.
- Measurement of major substrates and metabolites (glucose, glutamine, lactate, and glutamate) in culture medium using the YSI 2950 analyzer. This instrument is able to rapidly (~1 min per metabolite) determine the concentration of these important metabolism substrates from a small volume of sample (100-200 µL). Using a 96-well format, this analysis provides an economical method to roughly define changes in metabolism (e.g., in proliferating versus quiescent cells), and is also useful for checking cells in culture have sufficient supply of the major nutrients glucose and glutamine.
- The YSI 2950 metabolite analyzer is able to measure glucose, glutamine, lactate and glutamate in media in 2-3 minutes for all 4 metabolites in a 96-well format. The minimum volume needed is 100 μl A full 96-well plate maybe analyzed in 3-4 hours and results are typically returned the same or next day.
- The XFp Analyzer can measure respiration in most cell types, including primary cells, adherent cells, and suspension cells (with attachment). With a 6-well format, the XFp Miniplate is ideal for pairwise comparisons (in triplicate) and the analysis of precious samples, such as patient-derived and other rare samples. We mainly focus on the Cell Mito Stress Test to measure the key parameters of mitochondrial function: basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity. Other protocols using alternate substrates or permeabilized cells are available.
- The Shimadzu QP2010 gas chromatograph-mass spectrometer (GC-MS) is used for broader metabolic analyses including quantification and determination of stable isotope (13C, 15N) labeling rates of intra- and extracellular metabolites, including amino acids, keto and fatty acids, short-chain fatty acids, cholesterol, sugars, and sugar phosphates. Analysis of cells, tissues, tumors, feces, and serum or plasma can be performed. The GC-MS includes chemical ionization options, and sensitive methods using negative chemical ionization are under development.
- Scripps LC-MS/MS facility - (Center for Metabolomics and Mass Spectrometry) for broad profiling of metabolites.
For a complete list of services, please call (858)646-1000 ext. 3941 or email us.
|Cancer Metabolism Services||Internal
|Polar metabolite GCMS quantification (per sample)||$40||$50||$54||$105.20||CMR162|
|Polar metabolite 13C GCMS analysis (per sample)||$40||$50||$54||$105.20||CMR163|
|Fatty acid GCMS analysis (per sample)||$44||$55||$59.40||$115.72||CMR164|
|Cholesterol (sterol) GCMS quantification (per sample)||$40||$50||$54||$105.20||CMR165|
|Seahorse XFp (per plate)||$200||$250||$270||$526||CMR166|
|YSI metabolite analysis (per plate)||$66||$82.50||$89.10||$173.58||CMR167|
|Combined GCMS 13C & quantification polar metabolites (per sample)||$70||$87.50||$94.50||$184.10||CMR170|
|Fatty acid 13C GCMS analysis (per sample)||$40||$50||$54||$105.20||CMR171|
|Short-chain fatty acid GCMS quantification (per sample)||$44||$55||$59.40||$115.72||CMR172|
|Sugars GCMS analysis (per sample)||$40||$50||$54||$105.20||CMR173|
|Sample prep – cells (per sample)||$4||$5||$5.40||$10.52||CMR174|
|Sample prep – medium (per sample)||$2||$2.50||$2.70||$5.26||CMR175|
|Sample prep – tumor, tissue (per sample)||$12||$15||$16.20||$31.56||CMR176|
|Sample prep – fecal (per sample)||$20||$25||$27||$52.60||CMR177|
Sample prep charges (CMR174-177) are for sample extraction prior to GCMS, and are in addition to the GCMS charges. Samples can be submitted as extracts and in these cases the prep charges will not apply. Contact the core for extraction methods.
CMR166 for Seahorse XFp analysis includes the cost of plates and reagents.
The YSI analyzer (CMR167) measures glucose, lactate, glutamate and glutamine in culture medium samples in a 96-well plate format.
David Scott, Ph.D.
David Scott focuses on the development and application of methods for metabolomics, enhanced by the use of stable isotope substrates. Stable isotopes (mainly 13C) provide an added layer of information to metabolomic studies – not only is static pool information obtained, but it is also possible to infer the origins of metabolites and to track flux through metabolic pathways. Methods used include the analysis of small polar metabolites (mainly amino acids and carboxylic acids) and fatty acids by gas chromatography-mass spectrometry. Other techniques are being developed to analyze and quantify an expanded range of (labeled) metabolites. We have applied these methods in a study of the central carbon metabolism in Escherichia coli, and, in several papers, on melanoma metabolism. Investigations of melanoma cells showed the importance of glutamine metabolism under hypoxia, identified a novel route for the utilization of the carbon backbone of glutamine for fatty acid synthesis, and defined pathways for proline synthesis. Other recent work uses alternately labeled sugars to simultaneously track different inputs to glycans in normal cells and cell lines from patients with glycosylation defects.
Please call (858)646-3100 ext. 3941 or use the button below to send us an email.