Triglyceride Assay from Cells and Tissues: Difference between revisions

Line 16:

# Take 180 uL of the bottom layer into a fresh tube.

# Dry in fume hood overnight (or until completely dry)

# Add 50uL of '''Butanol Mixture'''

# If absorbance is going to be measured by cuvette, use non-bolded values. If you are using a plate reader use bolded values.

# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample.

# Add 50uL '''(500uL)''' of '''Butanol Mixture'''

# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:

## Resuspend triglyceride and glycerol reagent with water if necessary.

## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).

## Prepare reagent, you need 560 uL '''(80uL)''' of glycerol reagent and 140 uL '''(20uL)''' of triglyceride reagent. Make a bit extra and combine in a falcon tube.

## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.

## 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).

## 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.

@@ Line 16: / Line 16: @@
 # Take 180 uL of the bottom layer into a fresh tube.
 # Dry in fume hood overnight (or until completely dry)
-# Add 50uL of '''Butanol Mixture'''
+# If absorbance is going to be measured by cuvette, use non-bolded values.  If you are using a plate reader use bolded values.
-# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample.
+# Add 50uL '''(500uL)''' of '''Butanol Mixture'''
+# Measure triglyceride levels using Sigma Diagnostic Kit using 5 uL of sample:
+## Resuspend triglyceride and glycerol reagent with water if necessary.
+## Calculate how many samples you have (samples + buffer blank + 6 standard curve values).
+## Prepare reagent, you need 560 uL '''(80uL)''' of glycerol reagent and 140 uL '''(20uL)''' of triglyceride reagent.  Make a bit extra and combine in a falcon tube.
+## Aliquot 700 uL into a cuvette or '''100 uL into a well of a 96 well plate'''.
+## 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).
+## 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.