Tr22421, by Fujifilm - Product details

1

Metabolic profiling and atherosclerosis quantification

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Serum metabolites (cholesterol, triglycerides, glucose, free fatty
acids) were assayed from blood obtained from mice after 6-hour fast per
manufacturer’s protocols (Thermo Scientific TR13421, TR22421, TR15408,
and Wako Diagnostics HR Series NEFA-HR(2)).
For glucose tolerance test, mice were fasted for 6 hours, injected with
10% D-glucose (1 g/kg), and tail vein blood obtained at different time points
was analyzed using a glucometer (Contour, Bayer Healthcare, Mishawaka, IN).
Serum L-amino acid were measured from mice fed standard or high protein
Western diets for 2 month or from fasted mice given an oral protein gavage for
the indicated time points using a colorimetric L-amino acid assay kit (Abcam,
ab65347) following the manufacturer’s protocol.
Quantification of atherosclerosis at the aortic root was as
follows^{12 (link),21 (link)}. PBS-perfused hearts were placed in a
cryostat mold containing tissue freezing medium. 10 μm thick sections
were taken from the samples beginning just caudal to the aortic sinus and
extending into the proximal aorta. Slides were fixed with 4% paraformaldehyde
and stained with Oil Red O. Images were taken by EVOS XL Core Cell Imaging
system and Oil Red O positive regions were quantified using ZEN microscope
software (Carl Zeiss AG).

Zhang X., Sergin I., Evans T.D., Jeong S.J., Rodriguez-Velez A., Kapoor D., Chen S., Song E., Holloway K.B., Crowley J.R., Epelman S., Weihl C.C., Diwan A., Fan D., Mittendorfer B., Stitziel N.O., Schilling J.D., Lodhi I.J, & Razani B. (2020). High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy. Nature metabolism, 2(1), 110-125.

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2

Zebrafish Metabolic Biomarker Assessment

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Blood collection was performed by inserting a glass capillary needle into the zebrafish’s dorsal aorta, as reported.⁽¹⁸⁾ Zebrafish blood was obtained by using a heparinized needle for blood collection along the body axis and posterior to the anus in the region of the dorsal aorta. Blood was collected from zebrafish after a 20‐hour fasting period. Blood glucose level was measured with Bayer Contour NEXT Diabetes EZ meter (Bayer AG, Germany) using Contour NEXT Blood Glucose Test Strips. Blood samples were diluted 1:10 in phosphate‐buffered saline. After centrifugation, the supernatant containing serum was collected. Triglyceride (TG), total cholesterol (TC), and alanine aminotransferase (ALT) levels in diluted serum were measured with kits according to the manufacturer’s protocol (Thermo Scientific #TR22421 for TG; Wako #439‐17501 for TC; Pointe Scientific #A7526 for ALT).

Park K., Gooz M., Ye Z., Zhang J., Beeson G.C., Rockey D.C, & Kim S. (2021). Flavin Adenine Dinucleotide Depletion Caused by electron transfer flavoprotein subunit alpha Haploinsufficiency Leads to Hepatic Steatosis and Injury in Zebrafish. Hepatology Communications, 5(6), 976-991.

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3

Quantifying Plasma and Hepatic Metabolites

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Colorimetric and fluorometric assays were used to measure metabolites in fasting plasma. Fasting plasma levels of total triglyceride (Thermo Scientific, TR22421), non-esterified fatty acids (Fujifilm Wako, 999-34691), cholesterol (Sigma Aldrich, A12216), and β-hydroxybutyrate (Cayman Chemicals, NC9044966) were measured following the manufacturer’s instructions. All assays utilized a standard curve with a known standard to determine concentration of specific metabolite. Extraction of hepatic triglyceride (TG) have been previously described^{22 (link)}. In brief, liver tissue was digested in KOH solution at 55 °C for 4 h. Lipids were then extracted from digested tissue via incubation with H₂O:EtOH (1:1 ratio) solution. Extracted lipids were then used in previously mentioned assays to determine hepatic concentration which were normalized to tissue mass. Hepatic cholesterol was extracted using 1X RIPA buffer (10 mM Tris–Cl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, and 140 mM NaCl).

Burr S.D., Chen Y., Hartley C.P., Zhao X, & Liu J. (2023). Replacement of saturated fatty acids with linoleic acid in western diet attenuates atherosclerosis in a mouse model with inducible ablation of hepatic LDL receptor. Scientific Reports, 13, 16832.

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4

Metabolic profiling and atherosclerosis quantification

Cited 2 times

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Serum metabolites (cholesterol, triglycerides, glucose, free fatty
acids) were assayed from blood obtained from mice after 6-hour fast per
manufacturer’s protocols (Thermo Scientific TR13421, TR22421, TR15408,
and Wako Diagnostics HR Series NEFA-HR(2)).
For glucose tolerance test, mice were fasted for 6 hours, injected with
10% D-glucose (1 g/kg), and tail vein blood obtained at different time points
was analyzed using a glucometer (Contour, Bayer Healthcare, Mishawaka, IN).
Serum L-amino acid were measured from mice fed standard or high protein
Western diets for 2 month or from fasted mice given an oral protein gavage for
the indicated time points using a colorimetric L-amino acid assay kit (Abcam,
ab65347) following the manufacturer’s protocol.
Quantification of atherosclerosis at the aortic root was as
follows^{12 (link),21 (link)}. PBS-perfused hearts were placed in a
cryostat mold containing tissue freezing medium. 10 μm thick sections
were taken from the samples beginning just caudal to the aortic sinus and
extending into the proximal aorta. Slides were fixed with 4% paraformaldehyde
and stained with Oil Red O. Images were taken by EVOS XL Core Cell Imaging
system and Oil Red O positive regions were quantified using ZEN microscope
software (Carl Zeiss AG).

Zhang X., Sergin I., Evans T.D., Jeong S.J., Rodriguez-Velez A., Kapoor D., Chen S., Song E., Holloway K.B., Crowley J.R., Epelman S., Weihl C.C., Diwan A., Fan D., Mittendorfer B., Stitziel N.O., Schilling J.D., Lodhi I.J, & Razani B. (2020). High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy. Nature metabolism, 2(1), 110-125.

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5

Metabolic Profiling in Mice

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Glycemia was measured on fresh blood samples from the tail vein using a glucometer (Roche, Accu-Chek Performa). Plasma was prepared from blood samples collected using syringes conditioned with EDTA and stored at −80 °C. Plasma hormones and metabolites were measured according to the manufacturer's instructions using the following commercial kits: Insulin (Millipore Sigma, SRI-13K), Cholesterol (Randox laboratories, CH200), Triglycerides (Thermo Fisher Scientific, TR22421) and NEFAs (Wako, 999–34691, 995–34791, 991–34891, 993–35191, and 276–76491).

Colas C., Mouchiroud M., Al Dow M., Kolnohuz A., Gélinas Y., Caron A, & Laplante M. (2022). DEPTOR loss impairs brown adipocyte development in vitro but has limited impacts in mice. Molecular Metabolism, 67, 101660.

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6

Comprehensive Metabolic Profiling in Liver Disease

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Fasting blood glucose was measured using a GLUCOCARD Vital glucometer (Arkay, MN USA). ELISAs measuring serum levels of Leptin (Crystal Chem 90,030), Adiponectin (Abcam ab108785), Insulin (ALPCO 80-INSMR-CH01), Transferrin (Abcam ab157724), Ferritin (Abcam, ab157713), Albumin (Abcam ab108791), and Hepcidin (Intrinsic LifeScience SKU# HMC-001) were quantified according to manufacturer’s protocol. Colorimetric assays measuring hepatic ALT activity (Sigma-Aldrich MAK052), serum triglycerides (ThermoFisher Scientific TR22421), and serum free fatty acids (Wako NEFA-HR (2)) were quantified according to manufacturer’s protocol. Hepatic iron content was quantified using an iron assay kit (Abcam ab83366), and hepatic triglycerides were quantified using the Non-Esterified Fatty Acid detection kit (Wako NEFA-HR (2)). All assays were performed in duplicate, and measured on a BioTek SYNERGY H1 microplate reader. A portion of liver was fixed in 10% formalin, embedded in paraffin, cut into 5 μm sections, and stained with Perls’ Prussian blue. Images were taken on a Zeiss Axioplan 2 light microscope.

Miranda M.A., St Pierre C.L., Macias-Velasco J.F., Nguyen H.A., Schmidt H., Agnello L.T., Wayhart J.P, & Lawson H.A. (2019). Dietary iron interacts with genetic background to influence glucose homeostasis. Nutrition & Metabolism, 16, 13.

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7

Hepatic Lipid Quantification Protocol

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Frozen liver samples were weighed (50–100 mg) and homogenized in ice-cold PBS (Gibco, 14190-051). Then, hepatic lipids were solubilized in 1% sodium deoxycholate and using the commercially available kits, hepatic triglyceride (Thermo Scientific, TR22421) and NEFA (Fuji Film) were quantified according to the manufacturer’s directions.

Habibi M., Ferguson D., Eichler S.J., Chan M.M., LaPoint A., Shew T.M., He M., Lutkewitte A.J., Schilling J.D., Cho K.Y., Patti G.J, & Finck B.N. (2023). Mitochondrial Pyruvate Carrier Inhibition Attenuates Hepatic Stellate Cell Activation and Liver Injury in a Mouse Model of Metabolic Dysfunction-associated Steatotic Liver Disease. bioRxiv.

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8

Metabolic Markers in Murine Serum

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Blood was acquired from submandibular bleed for fasting serum measurements. Blood glucose measurements were performed in duplicate using two CVS Health Advanced glucometers per collection, and the mean was taken. If the variance exceeded 10%, a third reading was noted and the outlier was removed. Nonesterified fatty acids (NEFA; Fujifilm, HR Series NEFA-HR2), triacylglycerols (TAG; Thermo Scientific, TR22421), and cholesterol (Fujifilm, 999–02601 and 993–02501) serum measurements were made using colorimetric assays following the manufacturer's instructions. ELISAs were used to measure serum insulin (Millipore Sigma, EZRMI-13K) and glucagon (Mercodia, 10–1271–01). All colorimetric assays were quantified using a Biotek Synergy HTX plate reader. HOMA-IR values were calculated as (Blood glucose (mg/dL) X Insulin (μU/mL)]/405) [21 (link)].

Stagg D.B., Gillingham J.R., Nelson A.B., Lengfeld J.E., d’Avignon D.A., Puchalska P, & Crawford P.A. (2021). Diminished ketone interconversion, hepatic TCA cycle flux, and glucose production in D-β-hydroxybutyrate dehydrogenase hepatocyte-deficient mice. Molecular Metabolism, 53, 101269.

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Tr22421

Lab products found in correlation

8 protocols using tr22421

Metabolic profiling and atherosclerosis quantification

Zebrafish Metabolic Biomarker Assessment

Quantifying Plasma and Hepatic Metabolites

Metabolic profiling and atherosclerosis quantification

Metabolic Profiling in Mice

Comprehensive Metabolic Profiling in Liver Disease

Hepatic Lipid Quantification Protocol

Metabolic Markers in Murine Serum

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