Biology Reference
In-Depth Information
for molecular brightness analysis of receptor-receptor interactions. Fluorescent li-
gands have been used to label receptors for monitoring diffusion and binding kinetics
(
Briddon et al., 2010
) but would require very slow dissociation kinetics in order to be
useful for molecular brightness analysis. Alternatively, a monoclonal, monovalent
Fab, recognizing the native conformation of the receptor, could be used provided
each Fab has exactly one fluorescent tag.
10.2.1.4
Plating cells
Seed cells at 5
10
5
cells per 25 mm coverslip or MatTek dish (coated with appro-
priate substrate) 12-18 h prior to transfection or following electroporation (40% con-
fluence). Note that the type of substrate will affect cell attachment and overall cell
shape, which has important implications for making FCS recordings from the plasma
membrane.
10.2.1.5
Transfecting cells
Lipofectamine, calcium phosphate, and electroporation are common transfection
methods. End the transfection in phenol red-free culture medium, as phenol has auto-
fluorescence. Wait 24 h prior to the start of the FCS experiment to allow time for the
receptors to reach the plasma membrane. Fluorescent receptors in the ER/Golgi or in
vesicles can enter the observation volume complicating interpretation of plasma
membrane FCS results. If the endogenous ligand for the chosen receptor is present
in serum, use media with dialyzed serum to minimize receptor internalization.
10.2.2
Instrument setup
10.2.2.1
Environment
The room should be maintained at constant temperature during FCS recording (68-
70
F). Detector sensitivity can decrease at high room temperatures, and changes in
temperature surrounding the sample can affect diffusion time.
10.2.2.2
Imaging setup
Turn on the instrument, allow 30 min for the laser to stabilize, and establish the
proper settings for the fluorescent probe. Choose the appropriate laser line, dichroic
mirror, and set the appropriate emission range to capture as much of the emission
spectrum as possible. Select the fastest scan speed using 12 bits with 514
514 res-
olution, high detector gain (1100), pinhole of 1 airy unit, and low laser intensity (1%)
to minimize photobleaching.
10.2.2.3
FCS setup
Choose the appropriate laser line, dichroic mirror or main bean splitter and set the
appropriate emission range to capture as much of the emission spectrum as possible.
Use a pinhole diameter of 1 airy unit and the lowest laser setting that gives good sig-
nal to noise with minimal photobleaching. Note that laser intensity will depend on the
