Serum BHB (Abcam ab83390, Abcam, Cambridge, MA, USA) and FFA levels (Roche Diagnostics GmbH, Mannheim, Germany) were measured by colorimetric assays. Serum hsCRP levels were measured with a high-sensitivity, particle-enhanced immune-turbidimetric assay (Roche Diagnostics).
Ab83390
Manufactured by Abcam
Sourced in United States, Austria
Ab83390 is a laboratory reagent for research use. It is a monoclonal antibody that targets a specific protein or antigen. The core function of this product is to facilitate the detection and analysis of the target molecule in various experimental settings. No further details on the intended use or specific applications are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab180875, by Abcam (5 mentions)
Cobas bio centrifugal analyzer, by Roche (1 mentions)
Echomri, by Echo Medical Systems (1 mentions)
Paraoxon, by Merck Group (1 mentions)
Labmaster system, by TSE Systems (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
Ysi 2300 stat, by Yellow Springs Instruments (1 mentions)
Fuji dry chem 7000v, by Fujifilm (1 mentions)
Microplate spectrophotometer, by Agilent Technologies (1 mentions)
Ab65348, by Abcam (1 mentions)
Glucose assay kit, by Abcam (1 mentions)
Sybr green pcr master mix, by Qiagen (1 mentions)
Nile red, by Merck Group (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Ethylenediaminetetraacetic acid, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Cytiva (1 mentions)
Hmscs, by American Type Culture Collection (1 mentions)
11 protocols using ab83390
1
Serum Biomarkers Measurement Protocol
2
Quantification of Acetoacetate and β-Hydroxybutyrate in Adipocytes
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Colorimetric acetoacetate (AcAc) assay kit (ab180875, Abcam, Austria) has been used to quantify the endogenous levels of AcAc in condition media of vehicle control, PUNI, NLCs-PUNI-KAA, NLCs-KAA, and orlistat treated adipocytes. In this non-enzymatic assay, an unknown sample containing AcAc was reacted with a substrate to generate a colored product after incubation for 10 to 15 min at 25 °C (protect the plate from light). The absorbance can be measured at 550 nm. The assay kit can detect samples containing acetoacetate as low as 25 µM. The reaction is specific for AcAc and does not interact with 3-β-hydroxybutyrate content.
The quantity of β-hydroxybutyrate (β-HB) in condition media was measured with an analyzer (Cobas-bio-centrifugal analyzer, Roche Diagnostics, Somerville, NJ, USA) with commercially established liquid reagent (ab83390, Abcam, Austria) containing β-HB dehydrogenase and nicotinamide-adenine dinucleotide as described by Williamson et al. [21 (link)]. During the assay, the β-HB dehydrogenase removes hydrogen molecule from nicotinamide-adenine dinucleotide and supply to β-HB. The amount of reduced nicotinamide-adenine dinucleotide was monitored by measuring the 340-nm absorption, which was directly proportional to the β-HB concentration in the sample.
The quantity of β-hydroxybutyrate (β-HB) in condition media was measured with an analyzer (Cobas-bio-centrifugal analyzer, Roche Diagnostics, Somerville, NJ, USA) with commercially established liquid reagent (ab83390, Abcam, Austria) containing β-HB dehydrogenase and nicotinamide-adenine dinucleotide as described by Williamson et al. [21 (link)]. During the assay, the β-HB dehydrogenase removes hydrogen molecule from nicotinamide-adenine dinucleotide and supply to β-HB. The amount of reduced nicotinamide-adenine dinucleotide was monitored by measuring the 340-nm absorption, which was directly proportional to the β-HB concentration in the sample.
3
Metabolic Assessment in Mice
4
Measuring Plasma Ketone Levels
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Blood glucose was measured using a commercial glucometer. Blood was collected from the facial vein of mice and plasma was isolated for subsequent biochemical parameters. Plasma β‐hydroxybutyrate (ab83390, Abcam, Cambridge, United Kingdom) and acetoacetate (ab180875, Abcam) concentrations were measured with commercially available kits. For cardiac ventricle tissue, the samples were deproteinated with 1 M perchloric acid according to the manufacturer’s instruction.
5
Metabolic Biomarker Profiling in Mice
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At the end of the study period, blood samples were procured after the mice had been fasted for 8 h and biochemical variables were measured using standard methods. Briefly, plasma glucose (glucose oxidase method by YSI 2300-STAT; Yellow Springs Instruments, Yellow Springs, OH, USA) and insulin levels were measured, and homeostatic model assessment of insulin resistance (HOMA-IR) values were calculated [33 (link)]. Lipids including triglycerides, high density lipoprotein (HDL)-cholesterol and low density lipoprotein (LDL)-cholesterol, very low density lipoprotein (VLDL), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) were also measured (Multiplex Assay kit [RADPK-81K], Millipore, Billerica, MA, USA), as were adiponectin concentrations (ELISA kits; Millipore). FGF21 and resistin levels were measured using mouse Quantikine ELISA kits (R&D Systems; Minneapolis, MN, USA). β-hydroxybutyrate was measured using colorimetric assay kits (ab83390; Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.
6
Metabolic Assessment in Mice
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Blood glucose levels were measured using a glucometer (Glutest Pro R). Serum insulin and free fatty acid (FFA) concentrations were measured using enzyme-linked immunosorbent assay (Morinaga, Yokohama, Japan) and enzymatic method (Wako, Osaka, Japan), respectively. Beta hydroxybutyrate (BHB) levels were determined using a colorimetric method (ab83390, Abcam). Serum total cholesterol, triglyceride (TG), and alanine aminotransferase (ALT) concentrations were measured using Fuji Dry-chem 7000 V (Fujifilm Corporation, Tokyo, Japan). For glucose tolerance tests (GTT), mice were fasted for 16 h with free access to water, followed by intraperitoneal glucose injection (2 g/kg). We measured blood glucose concentrations at 0, 15, 30, 60, and 120 min after injection. Immeasurable high glucose concentration (>600 mg/dl) was recorded at 600 mg/dl. All analyses except for GTT were performed in the ad libitum fed state.
7
Colorimetric Quantification of Ketone Bodies
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Cells (approximately 18 × 106) were collected by centrifugation (2000 g for 10 min at 4 °C). The cell pellet was resuspended in 1–2 mL of cold assay buffer. The cell suspension was sonicated 20× at bursts of 1 s and centrifuged at 10 000 g for 10 min at 4 °C. The supernatant was removed and stored on ice. After sample preparation, the colorimetric assay kits [β‐hydroxybutyrate (β‐HB), ab83390, Abcam; acetoacetate (AcAc), ab180875, Abcam] were used in accordance with the manufacturer’s instructions. The absorbance at 450 or 550 nm was recorded using a microplate spectrophotometer (BioTek).
8
Measuring Plasma and Cardiac Metabolites
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9
Quantification of Ketone Bodies and Glucose
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Ketone bodies (acetoacetate and beta-hydroxybutyrate), were analyzed using Abcam’s Acetoacetate (ab180875) and Beta-hydroxybutyrate (ab83390), respectively. The Acetoacetate kits provide a sensitive method to quantitate endogenous levels of AcAc in human blood. In this non-enzymatic assay, AcAc reacts with a substrate to generate a colored product that can be measured at 550 nm [27 (link)]. The reaction is specific for AcAc, and does not detect beta-hydroxybutyrate. The beta-hydroxybutyrate kits utilize beta HB dehydrogenase to generate a product that reacts with the colorimetric probe with an absorbance peak of 450 nm [28 (link)].
Serum from 30 diabetic and 28 non-diabetic mellitus patients were analyzed using Abcam’s glucose assay kit to quantify the amount of glucose in the blood. In this assay, glucose oxidase specifically oxidizes glucose to generate a product which reacts with a dye to generate color (570 nm). The generated color is proportional to the glucose amount.
Serum from 30 diabetic and 28 non-diabetic mellitus patients were analyzed using Abcam’s glucose assay kit to quantify the amount of glucose in the blood. In this assay, glucose oxidase specifically oxidizes glucose to generate a product which reacts with a dye to generate color (570 nm). The generated color is proportional to the glucose amount.
10
Differentiation of Human Mesenchymal Stem Cells
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Human mesenchymal stem cells (hMSCs) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). The cell culture agents, such as fetal bovine serum (FBS) and penicillin-streptomycin were obtained from Hyclone Laboratories, USA. Dulbecco’s modified eagle medium (DMEM), Ethylenediaminetetraacetic acid (EDTA), and trypsin were purchased from Gibco, Paisley, UK. The cell to cDNA synthesis kits and SYBR Green PCR Master Mix were obtained from Qiagen, Hilden, Germany. The assay kits were purchased commercially for β-hydroxybutyrate (ab83390, Abcam, Austria) and acetoacetate (ab180875, Abcam, Austria). The ELISA array-based protein assay kits were purchased from Qiagen (MEH004A, Qiagen, Hilden, Germany). Punicalagin (PUNI), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], Propidium iodide, Oil Red O, Nile red, JC-1 stain, 3-isobutyl-1-methyl-xanthine (IBMX), rosiglitazone, dexamethasone (DEX), human insulin, and all the other chemicals for molecular biology assays were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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