Biology Reference
In-Depth Information
serum, cells become largely devoid of LDs. (Note: this method works effectively in
cells like HeLa, 3T3, and 3Y1 cells, but not in others, e.g., those derived from he-
patocytes such as Huh7 and HepG2 cells.) Then the cells are transferred to the culture
medium containing 0.1 mM DHA in a complex with fatty acid-free bovine serum
albumin (BSA). The DHA-BSA complex is prepared by adding DHA to a fatty
acid-free BSA solution in phosphate-buffered saline (PBS) (
Brasaemle, Barber,
Kimmel, & Londos, 1997
).
13.1.2.2
Incorporation of newly synthesized TG into existing LDs
Cells are cultured in the LPDS-containing medium as described above and then in a
medium containing 0.4 mM oleic acid (OA)-BSA complex for 12 h to induce for-
mation of LDs of low electron density. Then the cells are transferred to a medium
containing 0.1 mM DHA-BSA and cultured for various periods of time.
The treatment with either DHA or OA gives rise to TG-rich LDs. In contrast,
by culturing cells in a medium containing free cholesterol bound with methyl-
-
cyclodextrin (MCD), LDs enriched with cholesterol ester (CE) are generated. The
cholesterol-MCD complex is prepared by adding cholesterol to the MCD solution
in a culture medium (e.g., Dulbecco's modified Eagle's medium) (
Christian,
Haynes, Phillips, & Rothblat, 1997
). By treating cells first with the 0.25 mM
MCD-cholesterol complex (corresponding to 50
b
g/ml cholesterol) and then
with DHA-BSA, incorporation of newly synthesized TG to CE-rich LDs can be
studied.
m
13.1.2.3
Sample preparation and objective measurement of the relative
electron density
Cells are treated in a fixative containing 2.5% glutaraldehyde, 2.4% formaldehyde,
and 1 mM calcium chloride in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at
room temperature. After rinses in 0.1 M sodium cacodylate buffer containing
1 mM calcium chloride, the cells are postfixed in a mixture of 1% osmium tetroxide,
0.1% potassium ferrocyanide, and 1 mM calcium chloride in 0.1 M sodium cacody-
late buffer (
White, Mazurkiewicz, & Barrnett, 1979
). Potassium ferrocyanide is
added to the solution to enhance contrast of the membrane. The specimen is then
dehydrated in a graded series of ethanol (e.g., 50%, 70%, 90%, 95%, and 100%),
stained en bloc in 1% uranyl acetate in 100% ethanol, substituted with propylene
oxide, and infiltrated with the Quetol812 resin mixture. After polymerization in
an oven at 60
C for 2 days, ultrathin sections are prepared and counterstained by
Reynold's lead citrate (
Reynolds, 1963
) before observation by TEM.
The electron density of an object in TEM images could vary depending on many
factors, but the relative electron density can be assessed objectively by taking two
internal reference points in each image: one is the specimen grid bar that does not
allow any transmission of electrons and gives the maximum electron density; the
other is the resin-only area, for example, extracellular space, that should be of min-
imum electron density (
Fig. 13.2
D).
