Media was sampled after cells were incubated in fresh media for 6 h. Known concentrations of U-13C internal standards (glucose, glutamine, and palmitate, Cambridge Isotopes) were spiked in media samples before extraction. Extraction was performed as previously reported.(Ivanisevic et al., 2013 (link)) Samples were measured by LC/MS analysis (see Supplemental Experimental Procedures ). The absolute concentrations were determined by calculating the ratio between the fully unlabeled peak from samples and the fully labeled peak from standards for each compound. The consumption rates were normalized by cell numbers.
Glucose
Manufactured by Cambridge Isotopes
Glucose is a common type of laboratory equipment used for the measurement and analysis of glucose levels in various samples. It serves as a core function for determining glucose concentration, which is essential in various scientific and medical applications.
Automatically generated - may contain errors
Lab products found in correlation
Palmitate, by Cambridge Isotopes (4 mentions)
Glutamine, by Cambridge Isotopes (3 mentions)
U 13c internal standards, by Cambridge Isotopes (3 mentions)
Lactate, by Cambridge Isotopes (3 mentions)
Glutamate, by Cambridge Isotopes (2 mentions)
Aspartic acid, by Cambridge Isotopes (1 mentions)
Bio gel p 2, by Bio-Rad (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
6 protocols using glucose
1
Isotope Labeling for Metabolite Quantification
2
Analyzing Metabolite Concentrations in Cell Media
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After incubating cells in fresh media for 24 hours, the spent media were collected and analyzed. Known concentrations of U-13C internal standards (glucose, lactate, glutamine, glutamate, and palmitate; Cambridge Isotopes) were spiked into media samples before extraction. Extractions were performed in glass to avoid plastic contamination as previously reported [21 (link)]. Samples were measured by LC/MS analysis, with the method described above. For each compound, the absolute concentrations were determined by calculating the ratio between the fully unlabeled peak from samples and the fully labeled peak from standards. The consumption rates were normalized by cell growth over the experimental time period.
3
Metabolite Quantification in Cell Culture
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After incubating cells in fresh media for 24 hr, the spent media were collected and analyzed. Known concentrations of isotope-labeled internal standards (glucose, lactate, glutamine, glutamate, palmitate, and aspartic acid; Cambridge Isotopes) were spiked into media samples before extraction. Extraction was performed with glass as previously reported (Yao et al., 2016b (link)). Samples were measured by LC/MS analysis, with the method described above. The absolute concentration of each compound was determined by calculating the ratio between the fully unlabeled peak from samples and the fully labeled peak from standards. The consumption rates (x) were normalized by cell growth over the experimental time period by using the following equation where N0 represents the starting cell number, t represents incubation time, DT represents doubling time, and Y represents nutrient utilization.
4
Metabolic Flux Analysis of Cell Culture Media
5
Isotopic Labeling of Recombinant Proteins in E. coli
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Protein expression was performed using E. coli BL21 (DE3) cells transformed with the required expression plasmid. A single colony was used to inoculate 10 ml of LB media supplemented with the appropriate antibiotic, before overnight incubation at 37 ºC. This was then used to inoculate 500 ml of 2xYT media, incubated at 37 ºC until an OD600 0.6-0.8 was obtained, at which point the cell culture was centrifuged at 5000 x g at 4 ºC for 20 minutes (200 ml per expression culture required). The resultant pellet was washed with 20 ml/ gram of pellet 5xM9 Salts solution without ammonium sulfate nitrogen source (33.9 g/L Na2HPO4, 15 g/L KH2PO4, 2.5 g/L NaCl). After washing the pellet was resuspended in 100 ml M9 minimal media.
Isotopically depleted M9 minimal media was supplemented with 12 C (99.9%)-glucose (Cambridge Isotope Laboratories) and 14 N (99.99%)-ammonium sulfate, as the sole carbon and nitrogen sources. The M9 minimal media cultures were further incubated at 37 ºC for 1 hour, before protein expression was induced with 1 or 0.1 mM IPTG as required, and incubated overnight at 18 ºC [27] . After incubation cultures were centrifuged at 5000 × g at 4 ºC for 30 minutes, and pellets were stored at -80 ºC until required.
Isotopically depleted M9 minimal media was supplemented with 12 C (99.9%)-glucose (Cambridge Isotope Laboratories) and 14 N (99.99%)-ammonium sulfate, as the sole carbon and nitrogen sources. The M9 minimal media cultures were further incubated at 37 ºC for 1 hour, before protein expression was induced with 1 or 0.1 mM IPTG as required, and incubated overnight at 18 ºC [27] . After incubation cultures were centrifuged at 5000 × g at 4 ºC for 30 minutes, and pellets were stored at -80 ºC until required.
6
Enzymatic Synthesis of Labeled Nucleotides
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Glucosamine (SdiLN): Materials. All non-stable isotope labeled nucleotides, nucleotidesugars, enzymes, and chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise stated. Ni-NTA Agarose was purchased from Qiagen (Santa Clarita, CA), and Bio-Gel P2 from Bio-Rad (Hercules, CA). Glucose ( 13 C6, 99%) was purchased from Cambridge Isotope Laboratories (Andover, MA). The enzymes used in the synthesis of SdiLN were prepared according published methods: GalE 60 , HP-39 61 , PmST1 62 . The UDP-Glucose ( 13 C6, 99%) was prepared by previously published methods 63 .
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