Biology Reference
In-Depth Information
2.1.5.1
Required materials
- HeLa cells; cells have to be maintained in DMEM containing 5% charcoal-
stripped fetal calf serum (FCS)
Luciferase-based reporter gene ((17-mer)
5x
-
b
Globin-Luc)
Gal-mRXR
a
, VP16-mRAR
a
, and VP16-cTR
a
receptor chimera expression
vectors
- Lysis buffer: 25 mM Tris-phosphate (pH 7.8), 2 mM EDTA, 1 mM DTT, 10%
glycerol, and 1% Triton X-100
- Luciferin buffer: 20 mM Tris-phosphate (pH 7.8), 1.07 mM MgCl
2
, 2.67 mM
MgSO
4
, 0.1 mM EDTA, 33.3 mM DTT
- Opaque white Optiplate-96-well plates (Perkin Elmer)
- MicroLumat LB96P luminometer (Berthold)
2.1.5.2
Protocol
2.1.5.2.1
Transient transfection of HeLa cells
10
5
cells are seeded per P24 well 2 h before the transfection with jetPEI reagent
(Ozyme, Saint-Quentin-en-Yvelines, France). Per well, add the plasmid mix
diluted in 50
m
L of 150 mM NaCl buffer. The mix contains 50 ng of Gal4
chimera (e.g., Gal-mRXR
a
w ld type(w)ormutant expression vectors
Gal-mRXR
a
Y402A), 40 ng of VP16 chimera (e.g., VP16-hRAR
a
or VP16-cTR
a
expression vectors), 200 ng of (17-mer)
5x
-
b
glob-luc, and 50 ng of cytomegalovi-
rus-
b
-galactosidase (CMV
b
gal; used as an internal control to normalize for vari-
ations in the transfection efficiency). The total quantity of DNA was adjusted at
1
m
g with pBluescript plasmid. Vortex gently and spin down briefly. Per well,
2
m
L of jetPEI is diluted in 150 mM NaCl buffer to a final volume of 50
m
L(N/
P
5). Vortex gently and spin down briefly. The 50
m
L jetPEI solution is added
to the 50
m
L DNA solution all at once (do not mix the solution in the reverse order).
Vortex-mix the solution immediately and spin down briefly. Incubate for 15-
20 min at room temperature. Add the 100
m
L jetPEI/DNA mixture dropwise onto
the cells in 1 mL of medium in each well and homogenize by gently swirling the
plate. The cells are incubated at 37
Cina5%CO
2
incubator for 24 h. Culture me-
dium is removed from the wells and replaced with fresh medium. The cells are in-
cubated at 37
Cina5%CO
2
incubator again for 24 h. Three wells per assay are
transfected to have triplicate measurements.
¼
2.1.5.2.2
Cell lysis
The cell lysis is performed with the 5
passive lysis buffer from Promega. A suf-
ficient quantity of 1
working concentration is prepared by adding 1 volume of
5
passive lysis buffer to 4 volumes of distilled water. The growth medium is re-
moved from the cells and a sufficient volume of phosphate saline buffer (PBS) is
gently applied to wash the surface of the well. Swirl briefly and completely remove
the PBS from the well. Dispense 100
m
Lof1
passive lysis buffer into each well.
The culture plate is placed on an orbital shaker with gentle rocking at room temper-
ature for 20 min. Then, the lysate is transferred to a 96-well plate.
