Biology Reference
In-Depth Information
8. Assay the lysed cells using a Dual Luciferase Reporter Assay Kit
according to the manufacturer's protocol and record the lumi-
nescence on a microplate reader. In short, for each well, dis-
pense the Luciferase Assay Reagent II (100
L), read fi refl y
luciferase activity with a measurement time of 10 s and a delay
time of 2 s, dispense the Stop & Glo Reagent (100
μ
L), read
Renilla
luciferase activity with a measurement time of 10 s and
a delay time of 2 s.
9. Calculate the ratio of
Renilla
to fi refl y luciferase expression for
each well to provide relative luciferase units (RLUs). The aver-
age and standard deviation for each of the triplicates is calcu-
lated from the RLU values.
μ
Transient transfection of the psiCHECK-miR122 reporter has
several limitations for high-throughput screening such as the high
cost of transfection reagents, extensive transfection procedures, and
increased variations between different plates and different days. To
create a robust high-throughput assay for small molecule inhibitors
of miR-122, a stable cell line that constitutively expresses a miR-
122 reporter system is necessary to reduce the number of manipula-
tions, to increase the density of wells screened per plate, and to
enhance the reproducibility of the assay. The following steps to cre-
ate the stable cell line can be applied not only to Huh7 cells but also
to any other cell line by adjusting the G418 selective pressure.
3.4 Determination
of the G418
Concentration for
Selection of Stable
Cells
1. Passage Huh7 cells into a 12-well plate and grow overnight at
37 °C until they reach 30 % confl uency.
2. Remove the growth media and treat the cells with DMEM
(1 mL) supplemented with G418 (0, 50, 100, 150, 200, 300,
400, 500, 600, 700, 800, or 1,000
g/mL).
3. Incubate the cells at 37 °C for 48 h. Visually inspect the cells
and note the concentration that causes ~80 % cell death after
48 h.
4. Continue treatment by replacing the media every 2-3 days
with freshly prepared selective media containing the same con-
centration of G418. Incubate the cells at 37 °C for 14 days
after the fi rst G418 treatment.
5. After 14 days, assay the cells for viability using a Cell Titer-Glo
Luminescent Cell Viability Assay according to the manufactur-
er's protocol and record the luminescence on a microplate
reader. In short, equilibrate the plate to room temperature for
approximately 30 min. Add a volume of Cell Titer-Glo Reagent
equal to the growth media volume. Mix for 2 min and allow the
plate to incubate at room temperature for 10 min. Record the
luminescence with a measurement time of 1 s. For Huh7 cells,
selection with 500
μ
g/mL G418 is suffi cient for 80 % cell death
after 48 h and 100 % cell death after 14 days (
see
Note 7
).
μ
