24-h urine collections obtained from mice housed in metabolic cages for 1–3 days (see ‘Metabolic Studies’ above) were used to assess daily NH4+ excretion, daily TA excretion, and daily urine pH. Since mice were sacrificed after each experimental duration for electrolyte analysis, there are larger sample sizes for day 1 than for day 2 or day 3 within the urinalysis data set (Figure 7 ). Urine NH4+ concentrations were assessed using a commercially available kit (AA0100, Sigma-Aldrich, St. Louis, MI) that was modified for use in 96-well plates, with samples diluted 1:100 and ran in triplicate. Daily NH4+ excretion rates were calculated using 24-h urine NH4+ concentrations and volumes. Titratable acid (TA) content was measured as described previously (Chan, 1972 (link); Lee et al., 2009 (link)). Briefly, 25 µL of 0.1 M HCl was added to 25 µL of urine, boiled for 2 min, and let cooled for 1 min. The volume of 0.4 M NaOH needed to bring the sample pH to 7.4 was quantified. Samples of distilled (DI) water were run in parallel. The total number of moles needed to bring samples to pH 7.4, less the moles needed to bring the DI water to pH 7.4, was multiplied by 24-h urine volumes to yield daily TA excretion. Urine pH was measured using a micro-pH electrode (Orion 9810BN, Thermo Fisher Scientific).
Aa0100
Manufactured by Merck Group
Sourced in United States
The AA0100 is a general-purpose laboratory equipment. It is designed to perform various tasks in a laboratory setting. The core function of this product is to assist researchers and scientists in their experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Mak006, by Merck Group (3 mentions)
Orion 9810bn, by Thermo Fisher Scientific (1 mentions)
Ab65340, by Abcam (1 mentions)
Urea assay kit, by BioChain (1 mentions)
Cytation 5 cell imaging, by Agilent Technologies (1 mentions)
1260 infinity, by Shimadzu (1 mentions)
Hpx 87h ion exclusion column, by Bio-Rad (1 mentions)
Elx800uv, by Agilent Technologies (1 mentions)
Ezrmi 13k, by Merck Group (1 mentions)
Du 600, by Beckman Coulter (1 mentions)
9 protocols using aa0100
1
Urinalysis of Mouse Metabolic Caging
2
Circadian Rhythm Metabolite Analysis
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3
Ammonia-Removal Efficacy of Intestinal Microbes
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To evaluate the ammonia-removal efficacy of selected intestinal microbes in vivo, NH4Cl was i.v. injected on the last day into the mice fed the symbiotic pair for 2 weeks at the dosage of 100 mg/kg of body weight to elevate blood ammonia (n=3 per group). The blood and fecal samples were collected at 1 h after injection of NH4Cl. The blood was centrifuged, and serum was collected for ammonia quantitation. After the sacrifice of animals, the brain and fecal samples were collected and homogenized. After homogenization, the samples were centrifuged and the supernatant was used for ammonia quantitation with an ammonia assay kit (AA0100; Sigma-Aldrich).
4
Urine and Serum Biomarker Quantification
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Urine and serum samples collected were stored at −80°C. Ammonia was measured using a commercially available kit (AA0100; Sigma St Louis, MO, USA). Urine creatinine was assessed using a colorimetric assay kit (ab65340; Abcam Cambridge, MA, USA). Urine and serum urea were measured using a urea assay kit (MAK006; Sigma).
5
Hepatic and Serum Urea and Ammonia Assay
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Hepatic and serum urea and ammonia levels were measured by commercial kits (MAK006 and AA0100, Sigma-Aldrich, Saint Louis, MO, USA). Liver tissues were homogenized in the urea assay buffer provided by the kit for urea assay, while livers were homogenized in the water for ammonia assay, followed by protein concentration measurement to normalize the samples. Serum samples were measured by the kits directly.
6
Measurement of Ammonia and Urea Levels
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Plasma and urine samples were diluted 1:10 and analysed for the measurement of ammonia levels using an ammonia assay kit. The manufacturer’s instructions were followed with small modifications (AA0100; Sigma-Aldrich, St. Louis, MO, USA). To analyse faecal samples, 50 mg mouse droppings were dissolved in 1000 µL of water and centrifuged at × g for 5min to remove cell debris. Then 20 μL of the supernatant was added to 200 μL of Ammonium Assay buffer and baseline absorbance was taken at 340 nm. Subsequently, 2 μL of glutamate dehydrogenase was added to each sample and incubated at room temperature for 10 min. The absorbance at 340 nm was then recorded. Ammonia levels were calculated as described in the protocol. For the urea assay, urine was diluted 1:50 fold and faecal samples were prepared as described above. Plasma was assayed directly using the urea assay kit (Z5030016; Bio chain, Newark, CA, USA).
7
Ammonia Quantification Assay Protocol
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Ammonia concentration was measured by an ammonia assay kit (Sigma-Aldrich, AA0100) according to the manufacturer’s protocol. Briefly, Cell medium was mixed with ammonia assay reagent and incubated for 5 min at room temperature. Measure the absorbance of each sample at 340 nm. 10 μl of L-Glutamate Dehydrogenase solution was added and incubated for 5 min at room temperature. L-Glutamate dehydrogenase reacts specifically with ammonia. Then, the absorbance was read at 340 nm in a Biotek Cytation5 cell imaging multi-mode reader. The decrease in absorbance at 340 nm is proportional to the ammonia concentration.
8
Organic Acids and Ammonia Analysis
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From the supernatant samples, the concentration of various organic acids was measured by HPLC (Agilent, 1260 Infinity), using a standard analytical system (Shimadzu, Kyoto, Japan) equipped with an Organic Acid Analysis column (Bio-Rad, HPX-87H ion exclusion column) at 35°C. The eluent was 5 mm sulfuric acid, used at a flow rate of 0.6 mL min-1 and compounds were detected by refractive index. A five-point calibration curve based on peak area was generated and used to calculate concentrations in the unknown samples. For determination of ammonia concentrations, we employed assay kits (Sigma, AA0100) according to the manufacturer’s protocols.
9
Comprehensive Biochemical Assays for Liver and Metabolic Function
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