Assessing isoproterenol-stimulated whole-body lipolysis in vivo: Difference between revisions
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== 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. | Note: Mice do not need to be in a fasted state prior to this test. | ||
Experimental protocol | == Experimental protocol == | ||
#Briefly anesthetize mice with isofluorane and collect blood via retro orbital bleed. Allow blood to clot over ice. | #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. | #Inject 10 mg/kg isoproterenol (prepared fresh in sterile PBS) via intraperitoneal injection. | ||
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#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively. | #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) | == 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 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. | #To remaining wells, add 2.5 uL of serum samples. | ||
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##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve. | ##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) | == 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 remaining wells, add 3 uL of serum samples. | #To remaining wells, add 3 uL of serum samples. | ||