Lipid Droplets and Viral Infections - Methods in Cell Biology - page 175

10.2.4.2 Chemical transfection
To transfect cells with DNA, our lab utilizes mainly X-tremeGENE 9 DNA (previ-
ously FuGENE6, Roche).

1. Cells are plated on day 0 so that they will be at 70% confluency on day 1.

Typically, 1-1.5 million Huh7 cells are plated in a 10 cm dish, or twice as much
in a 15 cm dish.
2. On day 1, cells are transfected. For a 15 cm dish, 10-15 m g of DNA is diluted in a
final volume of 600 m l of OptiMEM. Thirty to forty-five microliters of
X-tremeGENE 9 DNA Transfection Reagent (ratio transfection reagent ( m l):
DNA ( m g) 3:1) is added.

3. The Transfection Mixture is incubated for 20 min at room temperature, and
added dropwise to the cells.

¼

Half of these amounts are used for a transfection in a 10 cm dish.
A detailed manufacturer's protocol can be found at: https://www.roche-applied-
science.com/sis/transfection/tfn_docs/QuickP_X-tremeGENE%209%20DNA%
20Transfection%20Reagent.pdf .

10.2.5 Production of viral stocks and infection
All work that involves the manipulation, handling, culture, production, and concen-
tration of HCV, and DENV by research laboratories must be performed in accor-
dance with guidelines established by CDC/NIH. Laboratories working with
DENV must follow standard BSL-2 practices; laboratories working with HCV must
follow standard BSL-3 practices and procedures for growing and concentrating viral
particles. Please refer to your EH&S department for appropriate Biosafety proce-
dures and protocols.
For the production of HCV stocks:

1. Huh7.5 cells are electroporated with 10 m gof in vitro transcribed viral RNA as
described above.
2. Transfected cells are mixed with 20 ml of supplemented medium and incubated
overnight at 37 C.
3. The next day, the medium is replaced and the cells are incubated for fivemore days.
4. The medium containing the infectious virions is carefully taken off the cells and
filtered through a 0.45 m m filter.
5. FBS is added to the filtered viral stock to a final concentration of 20%.
6. The viral stocks are frozen in aliquots at 80 C.

For infection:

1. Huh7.5 cells are seeded at 80,000/well in 12-well plates at day 0.
2. On day 1, viral stocks are diluted 1:1 with medium without FBS (final
concentration of FBS ¼ 10%).
3. Virus is then added to cells in a total volume of 800 m l/well.