Assessing isoproterenol-stimulated whole-body lipolysis in vivo: Difference between revisions
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== Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) == | == Glycerol and Triglyceride determination from serum (use Sigma Diagnostics Triglyceride Assay Kit) == | ||
#To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard). | #To wells of a clear 96 well plate, add 3 uL of water (as blank), the appropriate volume of standard (the kit comes with a stock standard of 2.5 mg/mL triolein; to replicate wells, add 1, 2, 3 and 4 uL of this stock. The assay is only linear up to concentrations of 10 mg/mL, which is 4 uL of 2.5 ug/mL standard). | ||
#To | #To each well, add 80 uL of glycerol reagent. | ||
#To each well, add | #To each well, add 3-8 uL of serum samples (3 if you expect a lot of lipolysis, and greater volume if you expect less). | ||
#Incubate plate at 37 deg C for 5 minutes. | |||
#Read the plate at 540 nm. This is the initial reading. | #Read the plate at 540 nm. This is the initial reading. | ||
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present. | #To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present. | ||