Biology Reference
In-Depth Information
biochemical measurement of intracellular triglycerides and extracellular free fatty
acids is an easy and precise way to track the fat storage and utilization in cells.
10.2.3.1
Triglycerides extraction and enzymatic quantification
Infinity Triglycerides (Thermo Scientific) can be used for reliable assessment of
the triglyceride content in cells. This reagent, designed to measure triglycerides in
human serum, can be adapted for cell culture after proper extraction of triglycerides
from the cell.
10.2.3.2
Triglyceride extraction from cell culture
This protocol is for the triglyceride extraction of cells from one well of a six-well
plate.
1.
Cells are washed with 2 ml of PBS and subsequently incubated with 1 ml of
hexane:isopropanol (3:2) on a vertical shaker at 100 rpm, 3
10 min.
2.
The hexane:isopropanol is evaporated under nitrogen.
3.
Lipids are resuspended in 500
m
l of chloroform with 1% Triton X-100, and dried
again.
4.
Lipids are resuspended in 200
m
l of water, mixed, and quantified with Infinity
Triglycerides.
10.2.3.3
Triglyceride enzymatic quantification
The measurements are performed according to the manufacturer's protocol:
1.
20
m
l of the triglyceride solution are added to 200
m
l of the Infinity Triglyceride
solution and incubated at 37
C for 10 min.
2.
The absorbance at 490 nm is measured (see protocol from manufacturer for more
details).
We use serial dilution (1/2-1/32) of Glycerol Standard Solution (Sigma) to draw a
standard curve (1.41-0.0088 mM), allowing us to determine the concentration of our
sample. Experimental triplicates are recommended. To be in the linear range of the
test, few dilutions of the triglyceride solution can be tested prior to testing all the
samples.
10.2.3.4
Free fatty acid enzymatic quantification
Nonesterified fatty acids (NEFA) can be easily measured in the cell culture super-
natant with HR Series NEFA-HR(2) from Wako. For this assay, medium without
FBS should be used to avoid background from bovine fatty acids.
1.
After media are collected, 20
m
l is incubated with 100
m
l of Color Reagent A at
37
C for 10 min.
2.
50
m
l of Color Reagent B is then added and the solutions are incubated for another
10 min at 37
C.
3.
The absorbance at 550 nm is measured (see protocol from manufacturer for more
details).
