24
edits
Changes
Created page with "== Assessing Isoproterenol-Stimulated Whole-Body Lipolysis ''in vivo'' == ##Note: Mice do not need to be in a fasted state prior to this test. #Briefly anesthetize mice with ..."
== Assessing Isoproterenol-Stimulated Whole-Body Lipolysis ''in vivo'' ==
##Note: Mice do not need to be in a fasted state prior to this test.
#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.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.
NEFA determination from serum
#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 each well, add 100 uL of reaction buffer A
##Note: Mice do not need to be in a fasted state prior to this test.
#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.
#Wait 15 minutes.
#Briefly anesthetize mice with isoflurane and collect blood via retro orbital bleed. Allow blood to clot over ice.
#Centrifuge blood samples and collect serum.
#Assay serum for NEFA's and Glycerol/Triglycerides using Wako and Sigma assay kits, respectively.
NEFA determination from serum
#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 each well, add 100 uL of reaction buffer A