903
edits
Changes
updated volumes and added a new section describing tissue specific volumes to use
# Vortex vigorously then sit at room temperature for 5 min
# Centrifuge 10min at 13 000 RPM
# Take 180 200 uL of the bottom layer into a fresh tube. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
# Dry in fume hood overnight (or until completely dry)
# If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.
# Add 50uL '''(500uL)''' of '''Butanol Mixture'''. See [[#Suggested Volumes | Suggested Volumes]] for your specific tissue
# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
## Resuspend triglyceride and glycerol reagent with water if necessary.
## For standards add 0-5 uL of glycerol standard '''(or of a 1/10 dilution of the glycerol standard)'''.
## Add 5 uL of resuspended lipid to each well to start (also make a 5uL blank of the butanol mixture) and mix.
## Let sit for ~30 min at room temperature (or 5 min at 37C if you are in a hurry). If using > 10 uL of butanol mixture the solution may be cloudy, let it settle and it should become more clear.
## Measure absorbance at 540 nm.
## If any samples are A540<0.1 or above the 5uL standard A540 then repeat with more or less lipid as required.
===Suggested Volumes===
This is based on using a 96 well plate to measure final concentrations.
{| border="1"
|-
! Tissue/Condition !! Lysis Volume !! Chloroform Volume !! Resuspension Volume !! Assay Volume
|-
| Liver || 1 mL || 200 uL || 500 uL || 5 uL
|-
| Muscle || 1 mL || 200 uL || 50 uL || 5 uL
|}
