Blood glucose and blood lactate were measured via tail clip using the Accu-Check Aviva (Roche) and Lactate Plus meter (Nova Biomedical), respectively. Free fatty acids were quantified using a colorimetric enzymatic assay (Sigma-Aldrich), as instructed by the manufacturer. Insulin-like growth factor-1 was quantified using a colorimetric assay (R&D Systems), per the manufacturer.
Colorimetric enzymatic assay
Manufactured by Merck Group
Sourced in United States
Colorimetric enzymatic assay is a laboratory equipment used to measure the concentration of a specific analyte in a sample. It utilizes the catalytic properties of enzymes to induce a color change in the sample, which is then quantified using a spectrophotometer. The intensity of the color change is proportional to the concentration of the target analyte.
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Lab products found in correlation
5 protocols using colorimetric enzymatic assay
1
Quantifying Metabolic Markers in Mice
2
Biomarker Quantification in Blood
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Blood samples were collected in lithium-heparin, EDTA and serum tubes. Lithium-heparin tubes (4.5 mL LH PSTTM II, Becton-Dickinson, NJ, America) were centrifuged at 1300 CRF for 10 min at room temperature (RT), plasma was frozen at −80 °C until it was analyzed for glucose concentrations. Glucose was measured by means of an end-point technique (Siemens, The Netherlands). EDTA tubes (8 mL, Becton-Dickinson, NJ, America) were centrifuged at 1200 G for 15 min at 4 °C, and plasma was frozen at −80 degrees until it was analysed for free fatty acids concentrations. Free fatty acids were assessed using an enzymatic test kit according to the manufacturer’s protocol (InstruChemie, Delfzijl, The Netherlands). Serum tubes (5 mL, Becton-Dickinson, NJ, USA) were set aside for at least 30 min, where after they were centrifuged at 1300 G for 10 min at RT, serum was frozen at −80 degrees until it was analysed for ketone content and cortisol concentration. Beta-hydroxybutyrate (βHB) was determined via a colorimetric enzymatic assay (Sigma-Aldrich; St. Louis, MO, USA). Analysis was performed according to manufacturer’s protocol. Cortisol was measured with immunometric chemiluminescence (sandwich) assay with Immulite XPi (Siemens, Den Haag, The Netherlands).
3
Metabolite and Hormone Quantification
4
Determination of Liver Enzyme Activities
5
Colorimetric Assays for Lactate and Glucose
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Lactate concentrations were analyzed using a colorimetric enzymatic assay (Sigma-Aldrich). The lactate determination kit comes with premade lactate assay buffer, lactate probe, enzyme mix, and standards. Samples were prepared and analyzed as per the manufacturer's protocol. Briefly, CM samples were diluted 1:50 in lactate assay buffer. Sample (50 mL) was incubated with 50 mL reaction mixture for 30 minutes at RT. Absorbance was measured at 570 nm. Similarly, glucose concentrations were measured using an enzymatic assay as per the manufacturer's instructions (Abcam). Conditioned media were diluted 1:50 in glucose assay buffer and incubated with an equal volume of reaction buffer, at 37 C for 30 minutes. Absorbance was measured at 570 nm.
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