Ammonia assay kit

Rumen SCFA concentrations were determined using a gas chromatograph (HP6890N, Agilent Technologies, Wilmington, DE, United States) as described by Yang et al. (2012) (link). Rumen Ni concentration was determined in the supernatant of a 10 ml rumen fluid sample (centrifugation at 900 × g for 5 min) with the atomic absorption spectrometry fitted with a nickel hollow cathode lamp according to the method described by Rahnama and Najafi (2016) . Concentrations of L-lactate, D-lactate, and ammonia, and urease activity were determined by using the L-Lactate Assay Kit, D-Lactate Assay Kit, Ammonia Assay Kit, and Urease Activity Assay Kit (ab65331, ab83429, ab83360, and ab204697, Abcam Trading Co., Ltd., Shanghai, China), respectively, and a microplate reader (Infinite M200 PRO, Tecan Trading AG, Switzerland). SPSS V21.0 (SPSS Inc., Chicago, IL, United States) was applied for statistical comparison using the two-tailed t-test. Differences were considered significant when P < 0.05 in the two-tailed t-test.

Ammonia levels were measured in cell-conditioned medium in NSCLC harboring KRAS-G12C or KRAS-WT isoforms 48 hours after BEZ235 (25 nM) or BKM120 (1 μM) treatment, following the manufacturer's instruction (Ammonia assay kit, Abcam).

Blood was collected from euthanized mice by cardiac puncture. Blood was immediately centrifuged at 7500×g for 5 min at 4 °C, and the plasma was stored at − 80 °C until further analysis. The Ammonia Assay Kit (Abcam, Cambridge, MA) was used to determine plasma ammonia according to the manufacturer’s protocol.
Plasma amino acid concentrations were measured using ion-exchange chromatography on a High-Speed Amino Acid Analyzer L-8800/L-8800A (Hitachi). Plasma proteins were precipitated with an equal volume of 7% sulfosalicylic acid and centrifuged for 10 min at 13,000 rpm. 5 μl of 2.5 N LiOH and 10 μl stock internal standard (S-2-aminoethyl-L-cysteine hydrochloride) were then added to 100 μl of plasma supernatant before loading samples into the analyzer. Amino acids were quantified using L-8800 ASM software package.

Ammonia was detected using Ammonia Assay Kit (Abcam, ab83360). Cells (5 × 10⁵ per 60-mm plate) were plated in duplicates 18 h prior the experiment. After the treatment, cells were homogenized in the Assay Buffer to have final concentration 2 × 10⁶ cells/100 μl and centrifuged at 13000 × g for 10 min at 4 °C. 10 μl of supernatant was used for the analysis according to the manufacturer’s recommendations.

Myoblasts were seeded on Matrigel-coated 96-well white plates (human cells at 3000 cells/cm² and mouse cells at 10,000 cells/cm²). The next day, human myoblasts were switched to only 12.5 mM glucose-containing medium without pyruvate, glutamine, or serum for overnight. Mouse myoblasts were cultured in a serum-free medium with glucose, pyruvate, and glutamine until the point of the assay. Cells were treated with different combinations of Glucose (6.25, 12.5, and 25 mM), β-2-chloro-alanine (250 μM), etomoxir (40 μM), 2-DG (1, 5, and 10 mM), FIDAS V (1, 2, and 5 μM), or shMAT2A_1/shMAT2A_2 in complete medium overnight. For SAH and SAM measurements, ELISA Combo Kit (Cell Biolabs, San Diego, CA) was used; intracellular ATP levels were measured using a luminescent ATP detection assay kit (ab113849, Abcam, Cambridge, MA); and ammonia was measured using the ammonia Assay Kit (ab83360, Abcam, Cambridge, MA) according to the manufacturer’s instructions.

The total level of ammonia or ammonium ion was measured by using a commercially available Ammonia Assay kit (Abcam, UK). The chemical equation that drives the relationship between ammonia and ammonium is: $$\text{NH}3+\text{H}2\text{O}\leftrightarrow \text{NH}4++\text{OH}-$$
Ammonium chloride (NH4Cl) was used as standard. Samples were measured immediately on a colorimetric microplate reader at OD 570 nm. Concentration of ammonia and ammonium in nmol/μL (mM) in the test samples was calculated as: $$\text{Ammonia}\text{and}\text{ammonium}\text{concentration}=\text{BV}*\text{D}$$ where B is the amount of ammonia and ammonium in the sample well calculated from standard curve in nmol. V is the sample volume added in the sample wells (μL) and D is the sample dilution factor.