Adherent growing cells were seeded in 96-well flat bottom plates (TPP) at cell numbers determined for each cell line to reach semiconfluency after 3 days under the respective oxygen and low glucose conditions and to reach confluency after 5 days via preliminary testing. Thus, 250–350 cells per well, depending on the cell line, were seeded in 200 μl DMEM/10%FCS/Gentamycin/5 mM glucose. Cell plates were cultured for 5 days at 5% CO2 and 37 °C in humidified chambers at oxygen concentrations of 21 or 5% in hypoxia-incubators (Coy Laboratories Products Inc., Grass Lake, MI, USA) respectively. At least 3 independent wells per cell line were tested either with or without 3 mM 3-OHB or 1.5 mM acetoacetate (lithium salt) and LiCl (both Sigma-Aldrich) as control in parallel. Four independent experiments were performed with fresh cultured cell aliquots. At day 5, supernatants were removed for metabolite testing, 100 μl of fresh medium containing 5-bromo-2′-deoxyuridine (BrdU) were added for another 24 h, and cell proliferation rate was then analyzed by the BrdU test (Roche; Cell Proliferation ELISA) according to the manufacturer’s instructions.
Acetoacetate
Manufactured by Merck Group
Sourced in United States
Acetoacetate is a chemical compound that is commonly used in laboratory settings. It serves as a precursor for various chemical reactions and is an important intermediate in metabolic processes. Acetoacetate is a colorless, crystalline solid that is soluble in water and other polar solvents.
Automatically generated - may contain errors
Lab products found in correlation
β hydroxybutyrate, by Merck Group (2 mentions)
Lithium chloride (licl), by Merck Group (1 mentions)
Cell proliferation elisa, by Roche (1 mentions)
Dmem high glucose, by PAN Biotech (1 mentions)
Glutamine, by Merck Group (1 mentions)
Uridine, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by PAN Biotech (1 mentions)
E plate, by Agilent Technologies (1 mentions)
Cim plate, by Agilent Technologies (1 mentions)
Aminoguanidine hcl, by Merck Group (1 mentions)
Acquity uplc h class system, by Waters Corporation (1 mentions)
Acquity hss t3 column, by Waters Corporation (1 mentions)
D β hydroxybutyrate, by Merck Group (1 mentions)
Empower3 software suite, by Waters Corporation (1 mentions)
Qda mass detector, by Waters Corporation (1 mentions)
Urea, by Merck Group (1 mentions)
Pyrrole, by Merck Group (1 mentions)
Thiourea, by Merck Group (1 mentions)
Phenylhydrazine, by Merck Group (1 mentions)
Avance 400, by Bruker (1 mentions)
7 protocols using acetoacetate
1
Adherent Cell Proliferation Assay
2
Metabolic dysfunction in mitochondrial disease
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The SH-SY5Y neuronal control cells and mutant cybrid cells carrying m.3243A > G with 98%, 90%, and 70% mutation loads and 143B osteosarcoma cybrid control and mutant cells were cultured in standard DMEM high glucose media (4.5 g/L) or in low glucose (0.5 g/L) (PAN biotech, Aidenbach, Germany), supplemented with 10% foetal bovine serum (PAN biotech), 1% glutamine, and 50 μg/mL uridine (Sigma-Aldrich, Saint-Louis, MO, USA) at 37 °C in the presence of 5% CO2 as described elsewhere [16 (link)]. Control and mutant cells were exposed to 5 mM of acetoacetate and β-D-hydroxybutyrate (Sigma-Aldrich, Saint-Louis, MI, USA) in a low glucose medium for 48 h. Control cells were exposed to rotenone (200 nM) a complex I inhibitor for 15 h.
3
Measuring MCEC Proliferation and Migration
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Proliferation and migration of MCEC were measured real‐time using an xCELLigence device (Agilent) that monitors electrical impedance. For analysis of proliferation, 10,000 cells were added per well on an E‐plate (Agilent) and media contained 2% FCS and 10 mM acetoacetate, 10 mM β‐hydroxybutyrate (Sigma‐Aldrich) or the respective volumes of water or 1 M ethanol. Impedance was measured every 15 min for 24 h. Migration was analyzed using CIM‐plates (Agilent) with a pore size of 8 µm. 30,000 cells were added per well. Media in the upper chamber was serum‐free, while media in the lower chamber contained 2% FCS. 10 mM acetoacetate, 10 mM β‐hydroxybutyrate or the respective volumes of water or 1 M ethanol were added to the media in the upper and lower chamber. Electronic impedance in CIM plates was monitored every 15 min for a total of 24 h.
4
Systemic and Peripheral Methylglyoxal Administration
5
Quantitative Analysis of TCA Cycle Intermediates
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TCA cycle intermediates were quantified in MCEC treated with 2 mM acetoacetate or 2 mM D‐β‐hydroxybutyrate (Sigma‐Aldrich) or the respective controls for 24 h. Samples were extracted in methanol with sonication and the separation protocol was adapted from Uran et al (2007 (link)). In brief, samples were analyzed on an Acquity HSS T3 column (Waters) heated to 40°C connected to an Acquity H‐class UPLC system (Waters). The UPLC system was coupled to a QDa mass detector (Waters) in single ion record mode, using negative detector polarity and 0.8 kV capillary voltage. Data acquisition and processing was performed with the Empower3 software suite (Waters).
6
Synthesis and Characterization of Heterocyclic Compounds
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Aldehydes, acetophenones, urea, thiourea, phenylhydrazine, acetoacetate,
pyrrole, KOH, NaOH, ethanol, and 2-methyltetrahydrofurane were purchased
from Sigma-Aldrich and were used without further purification. NMR
spectra were recorded on a Bruker Avance 400 spectrometer, operating
at 400.13 MHz for 1H NMR and 100.62 MHz for 13C NMR. Chemical shifts (δ) are reported in parts per million
(ppm) relative to tetramethylsilane (TMS). High-resolution mass spectrometry
(HR-MS) analysis was carried out on a Bruker micrOTOF apparatus, equipped
with a selective electrospray ionization (ESI) detector.
pyrrole, KOH, NaOH, ethanol, and 2-methyltetrahydrofurane were purchased
from Sigma-Aldrich and were used without further purification. NMR
spectra were recorded on a Bruker Avance 400 spectrometer, operating
at 400.13 MHz for 1H NMR and 100.62 MHz for 13C NMR. Chemical shifts (δ) are reported in parts per million
(ppm) relative to tetramethylsilane (TMS). High-resolution mass spectrometry
(HR-MS) analysis was carried out on a Bruker micrOTOF apparatus, equipped
with a selective electrospray ionization (ESI) detector.
7
Colorectal Cancer Cell Culture and Manipulation
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Cell Culture Human CRC tissues were obtained with informed consent and in accordance with the ethical standards of the Changzheng Hospital Institutional Review Board. Primary CRC cells (CRC102, CRC105, and CRC108) and their liverspecific variants (CRC102-LM, CRC105-LM, and CRC108-LM) were maintained as previously described (Gao et al., 2013) . CRC105 cells have wild-type adenomatous polyposis coli (APC) and b-catenin, while CRC102 and CRC108 cells harbor mutated APC (codon 1197, CTC / TTT) and b-catenin (codon 41, ACC / GCC), respectively. When magnetic-activated cell sorting (MACS) was used to sort for CD110+ cells, dissociated tumor cells were incubated with a monoclonal CD110 antibody labeled with MicroBeads (Miltenyi Biotec) for 30 min at 4 C, followed by cleavage of the MicroBeads. For Lys deprivation, cells were cultured with Lysine-depleted DMEM (Thermo Scientific). Lysine, a-aminoadipate, a-ketoadipate, and acetoacetate were purchased from Sigma-Aldrich. For siRNA inhibition studies, cells were transfected with validated human AASS, HMGCL, LRP6, p300, c-myc, HBO1, and EZH2 siRNA or negative control siRNA (Ambion) at a final concentration of 100 nM in the presence of an Oligofectamine reagent (Invitrogen).
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