To measure intracellular triacylglycerol, cells were harvested and suspended in 5% NP-40 aqueous solution. Cell extracts were prepared with two cycles of heating (95 °C, 5 min) and cooling (on ice, 5 min) followed by a centrifugation at 12,000g for 5 min. Cellular triacylglycerol contents were determined with TG reagent (#T2449, Sigma) on a microplate reader (BMG Labtech) according to the manufacturer’s specifications.
Tg reagent
Manufactured by Merck Group
TG reagent is a laboratory product manufactured by Merck Group. It is a chemical reagent used for the quantitative determination of triglycerides in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by BMG Labtech (1 mentions)
Glucose god fs kit, by DiaSys (1 mentions)
Nefa c kit, by Fujifilm (1 mentions)
Ultrasensitive elisa kit, by ALPCO (1 mentions)
Free glycerol reagent, by Merck Group (1 mentions)
Accu check, by Roche (1 mentions)
Automatic biochemistry analyzer, by Hitachi (1 mentions)
3 protocols using tg reagent
1
Quantifying Intracellular Triacylglycerol
2
Retro-orbital Blood Sampling for Metabolic Assays
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Blood was collected in EDTA tubes through retro-orbital sampling. Except for tolerance tests (see above), blood was collected around 8 am. Mouse plasma glycerol was measured by enzymatic assay (free glycerol reagent, Sigma-Aldrich); plasma nonesterified fatty acid and triglyceride levels were measured using the NEFA C kit (Wako) and TG reagent (Sigma-Aldrich), respectively. Glucose levels were measured using a glucometer (AccuCheck, Roche) or Glucose GOD FS kit (DiaSys). Plasma insulin was measured using an ultrasensitive ELISA kit (ALPCO Diagnostics).
3
Comprehensive Metabolic and Hematological Analyses
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After 3 weeks of treatment, mice were euthanized to analyze the therapeutic efficacy. Blood samples were obtained from the tail vein and centrifuged at 2000g and 4°C for 30 min. Insulin and TG were measured using an insulin enzyme-linked immunosorbent assay (ELISA) kit (10-1247-01, Mercodia) and TG reagent (T2449, Sigma-Aldrich), which were calculated according to the manufacturer’s instructions. The number of red blood cells, white blood cells, and platelets (PLT) and the concentration of hemoglobin, hematocrit, mean corpuscular, mean corpuscular hemoglobin, PLT count, and plateletcrit were measured using a fully automatic hematology analyzer (Sysmex). Plasma alanine amino transferase, aspartate aminotransferase, albumin, creatine kinase isoenzymes (CK-MB), lactate dehydrogenase (LDH), blood urea nitrogen, and creatinine levels were measured using automatic biochemistry analyzer (Hitachi).
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