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== Assessing Isoproterenol-Stimulated Whole-Body Lipolysis ''in vivo'' Background ==Background#Lipolysis is the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis is induced through activation of beta adrenergic receptors. Isoproterenol/Isoprenaline is a non-selective beta adrenergic agonist structurally similar to adrenaline. By administering isoproterenol ''in vivo'', it is possible to artificially stimulate whole-body lipolysis and assess changes in the concentrations of the products of lipolysis (i.e., glycerol and free fatty acids) in the blood.
Note: Mice do not need to be in a fasted state prior to this test.
== Experimental protocol==
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.
== NEFA determination from serum (use Wako Diagnostics NEFA-HR(2) Microtiter Kit)==
#To wells of a clear 96 well plate, add 2.5 uL of water (as blank), appropriate standard (the kit comes with stock of 1 mEq/L. Dilute the stock in ddH20 to give 2.5 and 5 mEq/L standards. For the high standard, add 5 uL of the 1 mEq/L stock. Due to the extra volume, the concentration of this last standard is actually 1.97 mEq/L).
#To remaining wells, add 2.5 uL of serum samples.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.
== 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 wells, add 3 uL of serum samples.