LPS224, LPS246, T1000, SW872, HT1080, KP250 and 293T cells were tested to confirm they are mycoplasma negative, and cultured in DMEM (Life Technologies, 11965–084) containing 10% FBS (Gemini, 900–108). C2C12 myoblasts (ATCC, CRL-1772) were propagated in DMEM containing 20% FBS. To evaluate differentiation, myoblasts were grown to 80 to 90% confluence and switched to 2% horse serum (Life Technologies, 16050–122) in DMEM. For metabolic labelling assays, cells were maintained in glucose-free DMEM (Life Technologies, 11966–025) supplemented with 10% dialyzed FBS (Gemini, 100–108) and 10 mM [1, 2-13C]glucose (Sigma-Aldrich, 453188), or supplemented with 10% dialyzed FBS and 2 mM [3-13C]sodium pyruvate (Sigma-Aldrich, 490733).
1 2 13c glucose
Manufactured by Merck Group
[1,2-13C]glucose is a stable isotope-labeled compound used in research applications. It contains two carbon-13 atoms, one each at the first and second positions of the glucose molecule. This product is intended for use in scientific research, with specific applications determined by the researchers and their experimental protocols.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Mcf 10a, by Merck Group (1 mentions)
U 13c l glutamine, by Merck Group (1 mentions)
Mcf 7, by Merck Group (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Sodium l lactate, by Merck Group (1 mentions)
Mstfa, by Merck Group (1 mentions)
1 13c glucose, by Merck Group (1 mentions)
N methyl n trimethylsilyl trifluroacetamide, by Merck Group (1 mentions)
13c6 glucose, by Merck Group (1 mentions)
5 protocols using 1 2 13c glucose
1
Mycoplasma-Free Cell Culture for Metabolic Assays
2
Breast Cancer Cell Line Metabolism
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MCF 10A (ATCC CRL-10317), MCF7 (ATCC HTB-22) and MDA-MB-231 (ATCC HTB-26) cells were from the American Tissue Culture Collection (ATCC). MCF 10A cells are a non-tumorigenic breast cell line, MCF7 cells are a tumorigenic, luminal breast cancer cell line, and MDA-MB-231 cells are a metastatic, basal breast cancer cell line [30 (link)]. Dulbecco’s modified Eagle medium (DMEM) without glucose, glutamine, sodium pyruvate, and phenol red (Life Technologies) was used as the growth media. The growth media was supplemented to contain 5 mM glucose (Thermo Fisher), 3 mM glutamine (Life Technologies), 10% dialyzed fetal bovine serum (dFBS, Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (100X Penicillin-Streptomycin solution, Life Technologies). High-lactate cultures were supplemented with 10 mM sodium L-lactate (Sigma) for the MCF 10A cultures and 20 mM for the MCF7 and MDA-MB-231 cultures. The isotopic tracers were [1,2-13C] glucose, [U-13C] L-glutamine, and [U-13C] sodium lactate, all purchased from Sigma-Aldrich.
3
Metabolic Labeling with 13C-Glucose Isotopes
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The labeling substrates, [1-13C]glucose (99 atom%), [1,2-13C]glucose (99 atom%), and [13C6]glucose (99 atom%), were purchased from Sigma-Aldrich. Derivatization agents TBDMS [N-methyl-N-(t-butyldimethylsilyl) trifluoroacetamide plus 1% t-butyl-dimethylchlorosilane] and MSTFA [N-methyl-N-(trimethylsilyl) trifluroacetamide] were also purchased from Sigma-Aldrich. Medium components and other reagents were purchased from Himedia and Sigma-Aldrich.
4
Microbial γ-PGA Production via Labeled Glucose
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Strains and plasmids used in this study were provided in Table 1 . B. licheniformis WX-02 was acted as the original strain for constructing recombinants, and E. coli DH5α was served as the host strain for plasmid construction. B. licheniformis and E. coli were grown in LB medium with responsible antibiotic (20 μg/mL kanamycin, 100 μg/mL ampicillin, or 20 μg/mL tetracycline), when required.
The γ-PGA fermentation medium contains 20 g/L glucose, 5 g/L NH4NO3, 11.35 g/L Na2HPO4, 8.15 g/L KH2PO4, 0.20 g/L MgSO4⋅7H2O, 0.01 g/L MnSO4⋅H2O, 0.0008 g/L CaCl2, and 0.0045 g/L Na2-EDTA. B. licheniformis were grown in twenty-four well plates with 2 mL working volume, and cultivated at 220 rpm and 37°C for 14 h. For 13C-MFA, glucose was replaced by [1, 2-13C] glucose (Sigma-Aldrich, CAS#138079-87-5) in γ-PGA production medium.
The γ-PGA fermentation medium contains 20 g/L glucose, 5 g/L NH4NO3, 11.35 g/L Na2HPO4, 8.15 g/L KH2PO4, 0.20 g/L MgSO4⋅7H2O, 0.01 g/L MnSO4⋅H2O, 0.0008 g/L CaCl2, and 0.0045 g/L Na2-EDTA. B. licheniformis were grown in twenty-four well plates with 2 mL working volume, and cultivated at 220 rpm and 37°C for 14 h. For 13C-MFA, glucose was replaced by [1, 2-13C] glucose (Sigma-Aldrich, CAS#138079-87-5) in γ-PGA production medium.
5
Metabolic Tracing of TNBC Biopsies
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