Lipid Droplets and Viral Infections - Methods in Cell Biology - page 174

We use serial dilution (1/1-1/32) of the NEFA Standard Solution to draw a standard
curve (1-0.031 mM), allowing us to determine the concentration of our sample. Ex-
perimental triplicates are recommended. To be in the linear range of the test, a few
dilutions of the supernatants can be tested before testing all the samples.

10.2.3.5 Radiolabeling and analysis with TLC
1. For measuring TG synthesis rates, cells are incubated in the presence of
0.125 m Ci/ml [1- 14 C]oleic acid (GE Healthcare, 58 mCi/mmol) for 4 h.
2. [1- 14 C]Oleic acid is dried under a nitrogen stream and then complexed to 10%
bovine serum albumin before addition to the cells.
3. Lipids are extracted with hexane/isopropyl alcohol (3:2), and dried.
4. Lipids are loaded onto thin layer chromatography plates, and quantified with a
Bioscan AR-2000 instrument.

10.2.4 Cell transfection
10.2.4.1 Electroporation
To generate run-off transcripts of HCV genomes:

1. Plasmid DNA encoding HCV reporter viruses is linearized with PvuI and purified
by phenol-chloroform extraction.
2. After ethanol precipitation, DNA is dissolved in RNase-free water and used for
in vitro transcription reactions.

In vitro transcription is carried out using the MegaScript T7 kit (Ambion), according
to the manufacturer's protocol. Briefly,

1. 10 m g linearized DNA is transcribed in a 100 m l reaction for 4 h at 37 C.
2. DNA is digested by adding Turbo DNase I and incubated for 20 min at 37 C.
3. RNA is precipitated by adding 150 m l RNase-free water and 150 m l LiCl 2 , and
incubated at 20 C for at least 2 h.

4. RNA is purified by spinning down at 14,000 rpm for 20 min, washed with 70 %
ethanol, air-dried for 5 min, and resuspended in RNase-free water.

Hepatoma Huh7.5 or Huh7-Lunet cells are the only cells available thus far that can
support efficient replication of HCV in culture. Because of their large size (9.8 kb),
electroporation is required to transfect cells with viral RNAs such as HCV's.

1. Cells are trypsinized, washed once in OptiMEM (Invitrogen), and resuspended in
Cytomix buffer at 10 7 cells/ml.
2. 400 m l of the cell suspension is mixed with 10 m g HCV RNA in 0.4 cm cuvettes
(Gene Pulser) and pulsed at 260 V and 950 m F with the Gene Pulser II (Bio-Rad).
3. Cells are plated to a concentration of

60% confluency.

4. 18-24 h later, the medium is changed.
5. The cells are then split according to their confluency and the experiment
schedule.