Biology Reference
In-Depth Information
Table 7.3
Visualization of Lipid Droplet Dynamics using BODIPY Fatty Acids
1. Coat cover slips with collagen solution and place in a six-well plate
2. Seed 0.2
10 6 freshly prepared hepatocytes per well
3. Incubate cells in serum-free DMEM overnight
4. Incubate cells with labeling medium A containing 558/568 C 12 for at least 4 h
5. Setup microscope for imaging with 491 and 543 nm laser lines
6. Transfer cover slips onto a culture chamber placed on the microscope in an environment
chamber thermostated at 37 C and supplied with 5% CO 2
7. Find fields of interest and record stage location
8. Carefully rinse once with PBS without disturbing stage locations
9. Carefully add labeling media B (time 0)
10.
Immediately start image acquisition every 1 min over a period of 30 min with Z-slices of
0.5
m
m steps
the diameter of the cover slip should be suitable for the adaptor. Otherwise, a glass
bottom culture dish (e.g., MatTek dishes) can be used instead. To coat cover slips or
glass bottom dishes with collagen, pipette collagen solution (Sigma) into wells con-
taining the cover slip, let the solution sit briefly, then remove the collagen solution,
and rinse wells twice with PBS. Collagen solutions can be reused if kept sterile. Cells
are then maintained in hepatocyte culture media at 37 C in humidified air containing
5% CO 2 for at least 4 h to allow attachment of the cells to the collagen matrices.
7.2.3.2 Oleic acid and BODIPY fatty acid incubations of hepatocytes
It is recommended that endogenously preformed CLDs should be reduced as much as
possible by incubating cells grown on collagen-coated cover slips in serum-free
DMEM overnight. Even though preformed CLDs always exist in hepatocytes iso-
lated from wild-type mice, this step helps reduce the stored TG pool and reduce
the background.
To visualize preformed LDs using exogenous fatty acid source, the intracellular
TG storage is first augmented by incubations with OA for at least 4 h. OA is com-
plexed to BSA to avoid lipotoxicity induced by free fatty acids. To make a 20
OA/
BSA complex, dissolve 5 g of FA-free BSA in 50 ml DMEM and warm up the mix-
ture to 56 C in a water bath. Weigh out 106 mg OA in a glass beaker (100 ml) using
analytical balance and warm up at 56 C water bath for 2 min. This step should be
done immediately before adding BSA in order to minimize oxidation. Add BSA so-
lution to the warmed up OA and stir vigorously using a stirring bar for 2 min. The
solution should clear, an indication that OA has been complexed with BSA. Filter-
sterilize the solution while it is still hand-warm and store at 4 C.
Labeling of preformed LDs is performed by mixing the red fluorescent fatty acid
analogue, BODIPY 558/568 C 12 , during OA loading (labeling medium A). After 4 h
incubation, cells are washed with PBS and incubated with labeling media containing
the fluorescent green fatty acid analogue, BODIPY FL C 12 . For imaging and quan-
tification of nascent LD formation under real-time conditions, we use labeling media
B for this step since a quencher is included in the labeling medium B to absorb
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