Biology Reference
In-Depth Information
in different chain length, and both BODIPY-C
16
and BODIPY-C
12
are reported to
incorporate well into cells (
Thumser & Storch, 2007
). We have only used BOD-
IPY-C
12
since with the inclusion of the BODIPY head group the analogue mimics
the natural chain length of OA. BODIPY-C
12
is esterified into both phospholipids
and neutral lipids and labels the same LDs as those stained with conventional LD
dyes (BODIPY 493/503 and Nile Red), thus showing properties similar to natural
long-chain fatty acids (
Wang et al., 2010
).
We used primary hepatocytes in this protocol due to our specific need to compare
cells of different genetic backgrounds; however, this method can be easily adapted to
other cell types. When a new cell type is used, several control experiments should be
performed to ensure that the analogues are metabolized as would be expected from
native fatty acids. Controls should include: (1) confirmation of esterification into
phospholipids and neutral lipids by TLC; (2) monitoring dynamics of analogue up-
take and incorporation, preferably compare these with radiolabeled OA; (3) coloca-
lization with conventional neutral lipid dyes.
This method can also be used to study the localization of nascent CLD formation,
interactions of proteins, and nascent LDs immediately after fatty acid loading of cells.
However, these applications require either transient expression of fluorescently tagged
proteins, or immunostaining of a protein of interest. Immunofluorescence staining of
LD-associated proteins is challenging since lipids cannot be fixed by general fixatives.
Some recently published protocols addressed this problem (
DiDonato & Brasaemle,
2003; Ohsaki, Maeda, & Fujimoto, 2005
). Primary hepatocytes are known for being
difficult to transfect. Liposome-based transfection reagents, such as Lipofectamine
2000, are able to deliver plasmid DNAs into hepatocytes when the ratio of transfection
reagent DNA is optimized; however, the transfection efficiency is generally very low,
less than 15%. We found that a cell type-specific reagent, Targefect-Hepatocytes,
together with its enhancer Virofect (Targeting Systems, CA), provides a much higher
transfection efficiency (
Wang et al., 2010
).
CONCLUSION
We have provided detailed protocols and expected outcomes for the purification and
analysis of hepatic LDs, including CLDs and LLDs. We have also presented a cell
biological method for monitoring LD dynamics in hepatocytes. These protocols can
be extended to study liver LD metabolism under different metabolic states.
Acknowledgments
Supported by grants from the Canadian Institutes of Health Research and Natural Sciences and
Engineering Research Council of Canada. Huajin Wang is supported postdoctoral fellowship
from the Canadian Institutes of Health Research. Richard Lehner is an Alberta Innovates-
Health Solutions Scientist.
