Biology Reference
In-Depth Information
20.1.1
Expression of receptors in cell plasma membranes
at low density
We often use Chinese hamster ovary K1 (CHO-K1) cells and T24 cells (ECV304,
a subclone of T24 human bladder carcinoma) because their cell membranes are
flat and we can easily observe single molecules of receptors by total internal
reflection (TIRF) microscopy. If the cell membranes are exceptionally rough, the
fluorescent intensities of diffusing individual receptor spots vary significantly,
making quantitative analysis of the intensities difficult. These cells are cultured in
HAM's F12 medium (Gibco) supplemented with 10% fetal bovine serum.
Using Lipofectamine PLUS (Life Technologies), we transfect the cells with
cDNAs encoding membrane receptors. cDNA sequences of GPI-APs are placed
in the Epstein-Bar virus-based episomal vector pOsTet15T3—which carries
tetracycline-regulated expression units, a transactivator (rtTA2-M2), and a Tet op-
erator sequence (a Tet-on vector)—and expressed in the cells. cDNA sequences
of GPCRs are placed in pcDNA3
(Invitrogen). The cells that stably expressed
the molecules of interest are selected and cloned using 800
m
g/ml G418 (final
concentration).
These cells are sparsely seeded (4
þ
10
3
per coverslip) in a glass base dish
(35 mm
f
with a window of 12 mm
f
, 0.15 mm thick glass; Iwaki). The expression
levels of the GPI-APs are adjusted by finely controlling the concentration of
doxycycline added to the cell culture medium. Specifically, the cells are grown
for a day in the glass base dish and are then incubated with various concentrations
of doxycycline for a day and used for single-fluorescent-molecule imaging. The
doxycycline concentrations are optimized by measuring the spot number
densities of fluorophores attached to the expressed receptors in cell plasma mem-
branes. Typically, the optimized doxycycline concentrations are 1-5 ng/ml for
GPI-APs and 10-50 ng/ml for transmembrane (TM) proteins. The optimal number
density of receptors for single-molecule observation is 0.1-2.0 molecules/
m
m
2
or
500-10,000 molecules/cell.
20.1.2
High-efficiency fluorescent labeling of receptors
We usually use two methods to label membrane receptors with fluorophores. One
method is to label receptors with fluorophore-conjugated Fab fragments of the an-
tibody. These fragments are conjugated with fluorophores with an amine-reactive
succinimidyl ester according to manufacturer instructions. The dye/protein (D/P)
ratios of fluorophore-labeled Fab fragment are estimated with optical density
measurements. When a receptor is tagged with Fab conjugated to a fluorescent probe
and the D/P ratio is 1 on average, the number of fluorescent probes on a single
Fab generally follows a Poisson distribution, varying mostly between 0 and
3 (no probe/Fab
19%, and 3
probes/Fab
¼
7%). Therefore, whether a single fluorescent spot in a single image
represents two Fab molecules or one Fab molecule conjugated with two or more
¼
37%, 1 probe/Fab
¼
37%, 2 probes/Fab
¼
