Cardiomyocytes were isolated from the hearts of adult mice as described above, and the CaCl2 concentration in the stopping buffer was gradually increased to 1mM. Cardiomyocytes were then pelleted by centrifugation at 100g for 3min, and resuspended in DMEM (Corning #90-113-PB) supplemented with 5mM glucose, 4mM L-glutamine (Corning #1-030-RM), 0.1mM sodium pyruvate (Sigma-Aldrich #P8574), 0.2mM BSA-conjugated palmitate (Sigma-Aldrich #A7030; Sigma-Aldrich #P9767), 0.2mM carnitine (Sigma-Aldrich #C0283), pH = 7.434 (link). 1250 live cardiomyocytes/well were plated to a 96-well plate (Agilent #101085-004) coated with 50μg/mL laminin (ThermoFisher #23017-015) and allowed to attach for 1 hour in a CO2-free 37°C incubator. A Seahorse XF96 extracellular flux analyzer (Agilent) was used to measure oxygen consumption rate (OCR) at baseline and after sequential additions of 3μM oligomycin (Sigma-Aldrich #O4876); 1.5μM FCCP; and 2μM rotenone (Sigma-Aldrich #R8875) + 2μM antimycin A (Sigma-Aldrich # A8674). Basal, ATP-linked, non-mitochondrial, and maximal respiration; proton leak; and respiratory reserve capacity were calculated as described previously34 (link).
P8574
Manufactured by Merck Group
P8574 is a laboratory equipment item produced by Merck Group. It is designed for general laboratory use, but a detailed description of its core function cannot be provided without the risk of interpreting or extrapolating beyond the available factual information.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using p8574
1
Assessing Cardiomyocyte Mitochondrial Function
2
Glucose and Insulin Tolerance Tests
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Clenbuterol was not administered on the day of a test. Mice were fasted for 5 h (prior to GTT and ITT) or 12 h (prior to pyruvate tolerance test). For GTT, glucose 2.5 g/kg lean weight (unless stated otherwise) was either injected i.p. or orally gavaged. For ITT and pyruvate tolerance test, human insulin 1 U/kg body weight (Actrapid, NovoNordisk) or pyruvate 2.5 g/kg lean weight (P8574, Sigma Aldrich) was injected i.p. In OGTTs, IPGTTs and pyruvate tolerance test, the total AUC was calculated with 0 mmol/l blood glucose as a baseline. Please note that the effect on fasting blood glucose contributes to the effect on total AUC. During OGTTs, plasma insulin was measured at time points 0 and 15 min by Elisa kit (90080, CrystalChem). In ITT, the rate of blood glucose disposal (KITT) was calculated as 0.693 × 100/t1/2, where t1/2 is the time necessary to reduce blood glucose by half, calculated from the linear regression analysis of data at 0, 15 and 30 min [23 (link)]. Mice were accessed in random order.
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