RKO, SW480 human colorectal cancer cells, 293T human embryonic kidney cells, HeLa human cervical cancer cells, and C2C12 mouse skeletal myoblast cells were obtained from the Cell Bank of Chinese Academy of Sciences and maintained at 37°C in DMEM (Hyclone). For β‐hydroxybutyrate treatment, cells were cultured in DMEM containing 5.6 mM glucose and then treated with 5 mM β‐hydroxybutyrate (Sigma‐Aldrich) for 4 h.
β hydroxybutyrate
Manufactured by Merck Group
Sourced in United States
β-hydroxybutyrate is a laboratory equipment product used for the quantitative determination of β-hydroxybutyrate levels in biological samples. It serves as an analytical tool for researchers and clinicians to measure this metabolite, which is a key indicator of ketosis and related metabolic conditions.
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Lab products found in correlation
Insulin, by Crystal Chem (2 mentions)
Butyrate, by Merck Group (2 mentions)
Acetoacetate, by Merck Group (2 mentions)
Glutamate, by Merck Group (2 mentions)
Mannitol, by Merck Group (2 mentions)
Rotenone, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Malate, by Merck Group (2 mentions)
Succinate, by Merck Group (2 mentions)
Edta microvette, by Sarstedt (1 mentions)
Contour next, by Bayer (1 mentions)
Lactate scout 4 analyzer, by EKF Diagnostics (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Pyrrolidine dithiocarbamate, by Merck Group (1 mentions)
Cell culture flask, by Thermo Fisher Scientific (1 mentions)
Histone h3 antibody, by Abcam (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
20 protocols using β hydroxybutyrate
1
β-Hydroxybutyrate Treatment Across Cell Lines
2
Plasma Biomarker Analysis in Rodents
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Whole-blood samples were collected from the tail into EDTA-microvettes (Sarstedt Inc) and were then centrifuged at 1000g for 10 min at 4 °C. Supernatants were aliquoted and stored at −20 °C. Plasma insulin and glucagon were determined by ELISA (Crystal Chem). Plasma free fatty acids (FFAs) (Abcam) and β-hydroxybutyrate (Sigma-Aldrich) were measured using a commercial kit.
Blood glucose and lactate levels were measured using a glucometer (Bayer Contour Next, Bayer Healthcare) and a lactometer (Lactate Scout 4 analyzer, EKF Diagnostics) with tail-tip bleeding.
Blood glucose and lactate levels were measured using a glucometer (Bayer Contour Next, Bayer Healthcare) and a lactometer (Lactate Scout 4 analyzer, EKF Diagnostics) with tail-tip bleeding.
3
Dietary Beta-Hydroxybutyrate Regulates Lifespan
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β-Hydroxybutyrate (Sigma–Aldrich, St. Louis, MO, USA, working concentration, 2 mM) was added to the standard food media. Three-day-old esg > GFP, wild type flies, 38-day-old esg > GFP, wild type flies, and 3-day-old esg > GFP + Catn1, Catn1/+ flies were fed 2 mM β-HB in standard media for seven days at 25 °C [18 (link),22 (link)]. Every two days, flies were transferred to fresh food vials containing β-HB.
4
Trimetazidine Modulates Metabolic Pathways
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BTECs were treated with 500 uM trimetazidine (1-(2,3,4-Trimethoxybenzyl) piperazine dihydrochloride, 653322 Sigma-Aldrich, St. Louis, MO, USA) dissolved in Phosphate-Buffered Saline (PBS) 1× solution for 72 h. Afterward, the conditioned medium (CM) and cell lysate were collected and used for metabolic analysis or in LLC cells treatment. LLC cells were treated with increasing concentrations (i.e., 1, 5, 10 mM) of β-hydroxybutyrate (H6501 Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS 1× solution.
5
Investigating NF-κB Regulation in Cell Cultures
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Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium, trypsin (with EDTA), antibiotics (50 U/mL penicillin; 50 μg/mL streptomycin), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from HyClone Laboratories Inc. (Logan, UT, USA). PA, β-hydroxybutyrate, N-acetylcysteine (NAC, an antioxidant), and pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). p-IκBα, IκBα, NF-κB p65, and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and histone H3 antibody was purchased from Abcam (Cambridge, MA, USA). Cell culture flasks were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and six-well plates, 96-well plates, and filters were purchased from Nalge Nunc (Rochester, NY, USA).
6
Fasted Metabolic Biomarker Profiling
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Fed hormones/metabolites levels were determined by collecting tail blood from mice that were without food for 3 h. Fasted hormones/metabolites levels were assessed in mice provided only with water ad libitum and without food for the indicated period. Time at day at which blood was collected was the same between groups. Tail vein blood was assayed for glucose levels using a standard glucometer (Nova Biomedical). Plasma was collected by centrifugation (1000 xg) in EDTA-coated tubes (Kent Scientific) and assayed using the indicated commercially available kits: β-hydroxybutyrate (Sigma), insulin (Crystal Chem. Inc.), TNF-α (Biovision), IL1-β (Biovision) and IL-6 (Biovision), S100A9 (R&D) and NEFA (Wako Chemicals).
7
Comprehensive Blood Biomarker Analysis in Mice
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At the end of each experiment, blood samples were collected from the mice in EDTA-coated tubes. To measure blood count, fresh blood samples were analyzed on a hematology analyzer (ProCyte Dx; IDEXX Laboratories) and centrifuged at 500g for 15 min. The collected plasma concentration was used to determine leptin (Novus Biologicals, reference no. M0B00), adiponectin (Novus Biologicals, reference no. MRP300), insulin [U-PLEX Mouse Insulin assay (Meso Scale Discovery), reference no. K1526HK], IGF-1 (Novus Biologicals, reference no. MG100), growth hormone (Ozyme, reference no. MOFI00849), β-hydroxybutyrate (Sigma-Aldrich, reference no. MAK041), and triglyceride circulating levels (Sigma-Aldrich, reference no. MAK266) using enzymatic methods from commercially available kits. NEFAs were detected with an enzymatic colorimetric assay (Fujifilm; Wako). Total cholesterol, triglycerides, and creatinine were measured by IDEXX Laboratories.
8
Cecal Content and Bacterial Supernatant Organogenesis
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Cecal contents (100 mg) were diluted in 1 mL of serum-free DMEM/F12 (Gibco) medium and vortexed for 1 h. The contents were centrifuged at 4,000 rpm for 10 min and supernatants were passed through a 0.22-·m syringe filter (Pall Corp.) before cultivation. Bacterial culture supernatants were prepared by centrifuging at 12,000 rpm for 10 min and passing the supernatants through a 0.22-·m syringe filter (Pall Corp.). To address the effectiveness of cecal contents or bacteria culture supernatants in promoting organogenesis, we used ENR or WENR media supplemented with cecal content supernatant (0.01%) or bacterial culture supernatant (4%) diluted in advanced DMEM/F12. For inhibition of Gpr41/43, we used Gpr43 antagonist (GLPG0974; 0.1 µM, Tocris) and Gpr41 antagonist (β-hydroxybutyrate, 3 mM; Sigma).
9
Fetal Liver Slice Culture with Metabolites
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Fetal livers (E19.5) were sliced < 0.1-mm-thick using two adjacent scalpel blades and cultured on collagen-coated plates at 37°C for 24 hr in DMEM:F12 with 10% fetal bovine serum containing 5 mM butyrate, 5 mM β-hydroxybutyrate, or 0.3 μM trichostatin A. All of these chemicals were acquired from Sigma-Aldrich (Saint Louis, MO).
10
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.
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