Biology Reference
In-Depth Information
size of protein complexes. Molecular brightness analysis has been used to explore the
oligomeric status of nuclear retinoid X receptors (
Chen, Wei, & Muller, 2003
), epi-
dermal growth factor receptors (
Saffarian, Li, Elson, & Pike, 2007
), urokinase plas-
minogen activator receptors (
Malengo et al., 2008
), and biogenic amine receptors
(
Herrick-Davis, Grinde, Cowan, & Mazurkiewicz, 2013; Herrick-Davis, Grinde,
Lindsley, Cowan, & Mazurkiewicz, 2012
).
This chapter focuses on the application of FCS and PCH for monitoring receptor-
receptor interactions. The methods described are for a confocal microscope setup using
one photon excitation. Using a commercially available confocal microscope fully
equipped for FCS data collection and analysis is the most straightforward approach.
A homebuilt FCS system is an option for those who are well acquainted with biophys-
ical techniques (
M
ller et al., 2003
). The first commercial FCS instrument was intro-
duced in 1997 by Zeiss and called the ConfoCor 2 and, in 2000, was combined with a
Zeiss LSM 510 as a single integrated instrument platform (
Weisshart, Jungel, &
Briddon, 2004
). Since that time, all the major microscope manufacturers, Nikon,
Olympus, and Leica, offer FCS/LSM systems. The methods and data presented in this
chapter are based upon our experience with a Zeiss ConfoCor 3 avalanche photodiode
detection module mounted on an LSM-510 microscope and with the Zeiss LSM-780
microscope integrated with a gallium arsenide phosphide photon detector.
Ï‹
10.1
MATERIALS
1.
Confocal laser scanning microscope (Zeiss, Nikon, Lecia, Olympus)
The instrument should be equipped with a stable excitation source (argon ion,
HeNe laser), a 40
1.2 NA C-apochromat water immersion objective,
avalanche photodiode or gallium arsenide phosphide photon-counting detector,
and autocorrelation analysis software (Zeiss Aim or Zen; Origin; MATLAB).
2.
Cells
HEK293 or CHO for transfection or primary cultures expressing native
receptor of interest, along with the appropriate culture medium and culture
dishes for growing cells.
3.
Fluorescent tag
A GFP variant attached to the receptor (most common), a fluorescent ligand, or
a fluorescent monovalent antibody.
4.
Plasmids
Monomeric (CD-86) and dimeric (CD-28) plasma membrane proteins with
GFP or YFP attached to the C-terminus are good controls for molecular
brightness analysis of plasma membrane receptors. Monomeric GFP or YFP,
expressed in the cell cytoplasm, for adjusting the pinhole alignment prior to
the start of each experiment.
5.
Transfection reagents
Lipofectamine reagent, calcium phosphate, or electroporation.
6.
Chambers for plating cells
Use 25 mm glass coverslips (thickness no. 1.0 or 1.5) inserted into a six-well plate
or use 35-50 mmdishes with glass bottoms (MatTek). If using coverslips, obtain a
