Biology Reference
In-Depth Information
10 cm dish, to obtain stable expression of either WT LHCGR or LHCGR
S
,we
transfect cells with 12
g of either
Lhcgr
S
or WT
Lhcgr
plasmid DNA and for cell
lines that coexpress LHCGR
B
and LHCGR
S
with 12
m
g
Lhcgr
B
and 12
g
Lhcgr
S
as per manufacturer's instructions. Geneticin selection (with 1 mg/ml in
standard culture media) is begun 48 h posttransfection and we replace media contain-
ing Geneticin every 48 h thereafter. After 5-7 days, it is usual to observe large quan-
tities of cell death and be left with a few cells, which will form distinct colonies.
Following the initial cell death, we subsequently reduce Geneticin concentration
to 0.5 mg/ml. We select colonies, expand, and screen for receptor cell surface expres-
sion using flow cytometry. We utilize the N-terminal tags of the receptors for detec-
tion of cell surface receptor expression and fluorescent secondary Alexa 488 and
Alexa 647 antibodies for detection of FLAG and HA.11 primary antibodies, respec-
tively. When screening, we try to select for stable cell lines that are monoclonal with
cells forming a distinct single peak of mean fluorescence. If the receptor expression
level is not uniform among the cell population, further cell sorting can be conducted
to obtain a pure clone.
As the LHCGR primarily couples to G
m
m
a
s to mediate its physiological functions
(reviewed by
Menon &Menon, 2012
), we routinely use assays that measure the sec-
ond messenger cAMP. We utilize three methods for measuring ligand-induced
cAMP accumulation: the measurement of cAMP directly using EIA kits (Assay De-
signs), live cAMP kinetics using the GloSensor
™
technology (Promega), and a re-
porter gene system of the cAMP response element fused to firefly luciferase
(CRE-luc). As CRE-luc activity is the assay we routinely use, we will describe
our protocol for assessment of cAMP using the CRE-luc system:
1.
Plate HEK293 stably expressing WT LHCGR and LHCGR
S
or coexpressing
LHCGR
B
/LHCGR
S
cells into 96-well plates to ensure a confluency of
80-90%.
2.
The following day, transfect each well with 40 ng of
CRE-luc
plasmid and 5 ng
of
pRL-CMV
plasmid. We use 0.5
l/well Lipofectamine
®
2000 as a
m
l OptiMEM as a diluent for both plasmid DNA
and Lipofectamine
®
2000 and follow manufacturer's instructions for the
transfection procedure.
Note
: the cotransfection of
pRL-CMV
plasmid serves as a control for
transfection efficiency, which can be used for normalization of data.
3.
For experiments using single stable LHCGR
S
, transient transfection of
Lhcgr
B
is also needed. We add 100 ng/well of
Lhcgr
B
plasmid DNA into the
transfection mix containing
CRE-luc
and
pRL-CMV
plasmids and 100 ng/well
of empty
pcDNA3.1
plasmid for a transfection control into WT LHCGR
functional comparisons.
4.
The following day, remove media and replace with 100
transfection reagent and 2
25
m
l phenol red-free
DMEM containing 0 or 10 nM hCG.
Note
: it is important to use phenol red-free
media as phenol red has been demonstrated to impact on the degree of signal
detection with the luciferase assay.
m
