Standards for cysteine, asparagine, glutamine, tryptophan, ornithine, citrulline, putrescine, spermine, spermidine, sarcosine, taurine, hypotaurine, a mixture containing 17 AAs, an isotopic algal mixture of 16 13C,15N-labeled AAs, 13C4,15N2-asparagine, 13C4-asparagine, 13C5,15N2-glutamine, 13C3,15N-cysteine, and 13C11,15N2-tryptophan were all purchased from Sigma Aldrich (St. Louis, MO). d8-putrescine was purchased from CDN isotopes (Quebec, Canada). Pronase and carboxypeptidase Y were purchased from Calbiochem (Darmstadt, Germany). LC/MS-grade water, methanol, and acetonitrile (ACN) were purchased from Burdick and Jackson (Muskegon, MI). Formic acid and heptafluorobutyric acid (HFBA) were obtained from Sigma Aldrich. L-asparaginase was obtained from Lundbeck pharmaceuticals (Deerfield, IL). Medium for cell culture (RPMI 1640) and fetal bovine sera (FBS) were purchased from Thermo HyClone (Logan, Utah). The liquid chromatography column (Zorbax SB C-18; 3.0 × 100 mm, 1.8 μm particle size) was obtained from Agilent (Santa Clara, CA). All other chemicals were of analytical grade and obtained from Sigma.
13c3 15n cysteine
Manufactured by Merck Group
13C3,15N-cysteine is a stable isotope-labeled amino acid. It contains carbon-13 and nitrogen-15 isotopes. This product is intended for use in various analytical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Glutamine, by Merck Group (1 mentions)
Heptafluorobutyric acid hfba, by Merck Group (1 mentions)
Asparagine, by Merck Group (1 mentions)
Tryptophan, by Merck Group (1 mentions)
Putrescine, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
13c5 15n2 glutamine, by Merck Group (1 mentions)
Zorbax sb c18, by Agilent Technologies (1 mentions)
Hypersep c18 column, by Thermo Fisher Scientific (1 mentions)
Methane sulphonic acid, by Merck Group (1 mentions)
Cysteine, by Merck Group (1 mentions)
Pancreatic lipase, by Merck Group (1 mentions)
3 protocols using 13c3 15n cysteine
1
Comprehensive Amino Acid Quantification
2
Isolation and Culture of Primary Mouse Hepatocytes
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Hepatocytes
from male mice (10–11 weeks) were isolated using a two-step
perfusion procedure as previously described.40 (link) Cell aggregates and liver chunks were removed by filtration with
cells enrichment by Percoll density centrifugation. Trypan blue exclusion
suggested greater than 95% viability. Hepatocytes were plated at a
density of 0.5 × 106 cells/well in 12-well plates
precoated with type I collagen in Williams E medium containing 5%
fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% Glutamax,
0.3 mM ascorbic acid, and 100 nM dexamethasone.41 After 3 h, nonadherent cells were removed, and fresh Williams
E medium containing 1% ITS Premix Universal Culture Supplement,42 (link) 1% penicillin/streptomycin, 1% Glutamax, 0.3
mM ascorbic acid, and 100 nM dexamethasone was replaced. The cells
were treated with 0.1 μM to 1 mM sulfasalazine, 10 nM TCDD or
vehicle (DMSO) and incubated for 24, 48, and 120 h at 37 °C and
5% CO2. For tracer studies, preincubation medium was exchanged
after 3 h for custom cystine/cysteine-free medium (Gibco) supplemented
with 13C3,15N-cysteine (Sigma, final
concentration 0.3 mM) and cells were treated with 1 and 10 μM
sulfasalazine, 10 nM TCDD or vehicle (DMSO) for 48 h.
from male mice (10–11 weeks) were isolated using a two-step
perfusion procedure as previously described.40 (link) Cell aggregates and liver chunks were removed by filtration with
cells enrichment by Percoll density centrifugation. Trypan blue exclusion
suggested greater than 95% viability. Hepatocytes were plated at a
density of 0.5 × 106 cells/well in 12-well plates
precoated with type I collagen in Williams E medium containing 5%
fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% Glutamax,
0.3 mM ascorbic acid, and 100 nM dexamethasone.41 After 3 h, nonadherent cells were removed, and fresh Williams
E medium containing 1% ITS Premix Universal Culture Supplement,42 (link) 1% penicillin/streptomycin, 1% Glutamax, 0.3
mM ascorbic acid, and 100 nM dexamethasone was replaced. The cells
were treated with 0.1 μM to 1 mM sulfasalazine, 10 nM TCDD or
vehicle (DMSO) and incubated for 24, 48, and 120 h at 37 °C and
5% CO2. For tracer studies, preincubation medium was exchanged
after 3 h for custom cystine/cysteine-free medium (Gibco) supplemented
with 13C3,15N-cysteine (Sigma, final
concentration 0.3 mM) and cells were treated with 1 and 10 μM
sulfasalazine, 10 nM TCDD or vehicle (DMSO) for 48 h.
3
Quantifying Bioactive Nitro Fatty Acids
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All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) if not stated otherwise. All nitro fatty acids and internal standards NO2-[13C18]OA, NO2-[13C18]LA and [15N]O2-cLA were synthesized as previously [30] (link)–[32] (link). Pancreatic lipase, cysteine, [13C3,15N]cysteine and methanesulphonic acid were purchased from Sigma-Aldrich (L3126, C122009, 658057, 471356). Gastric juice artificial was purchased from Fisher Scientific Company (S76772). Strata NH2 (55 µm, 70A) columns were from Phenomenex (8B-S009-HCH). Hypersep C18 columns were purchased from Thermo Scientific (60108-305). Mass spectrometry quality solvents were purchased from Burdick and Jackson (Muskegon, MI, USA). Extra virgin olive oils and fresh olives were from Jaen, Spain, and came from three different types of cultivars: Arbequina, Frantoio and Picual [33] (link).
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