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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 remaining wellseach well, add 3 80 uL of serum samplesglycerol reagent. #To each well, add 80 3-8 uL of glycerol reagentserum samples (3 if you expect a lot of lipolysis, and greater volume if you expect less). #Incubate plate at 37 deg C for 30 5 minutes.
#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.