Biology Reference
In-Depth Information
Oligomerization
photodiodes (MPD). The emission light is split by a 515 nm dichroic mirror and
guided into one of the two MPDs where the light is filtered by a 440/45 or 525/
45 emission filter (Chroma). The photon arrival times are recorded by a PicoHarp
300 time-correlated single-photon counting system (PicoQuant), controlled by Sym-
PhoTime 64 1.5 software (PicoQuant).
The contribution of cross talk, the fluorescence of one type of dye detected in the
“other” detector, will give rise to false-positive cross-correlation. This artifact will
be prevented by using pulsed interleaved excitation (PIE) (
M¨ ller, Zaychikov,
Br¨uchle, & Lamb, 2005
). The absorbance spectra of most fluorophores are rela-
tively narrow, and therefore, it is possible to select a pair of dyes such that each
dye can be excited exclusively by a specific laser line. When two pulsed laser units
are emitting in an alternating mode at a switching time much faster than the resi-
dence time of the labeled receptor in the detection volume, one will obtain the fluo-
rescence photons of both dyes in different time periods. Here, a semi-PIE approach
is used where the CFP laser is pulsing in combination with a continuous wave YFP
laser. By time gating the detected fluorescence, the cross-talk photons from mTur-
quoise2 in the YFP detection channel can be omitted in the calculation of the auto-
and cross-correlation curves. YFP fluorescence in the CFP detection channel can be
neglected (
Fig. 11.1
).
11.2
METHODS
In the following section, a protocol is presented that can be used to measure dual-
color FCCS in living cells using line-scan acquisition. Here, the homodimerization
of mTq2- and sYFP2-labeled H1 receptors, produced in HeLa cells, is studied but the
protocols can easily be adapted to monitor hetero-oligomerization. Protocols that de-
scribe point FCCS and PCH measurements are published elsewhere (
Hink, in
preparation; Hink, de Vries, & Visser, 2011
;
Chapter 10
).
11.2.1
Growth and transfection of HeLa cells
HeLa cells are grown in DMEMmedium supplemented with GlutaMAX in a live cell
incubator. One day before transfection, the cells are transferred from the culture flask
to sterile circular coverslips and stored in a six-well container, at a confluency of
10
5
cells). Six hours after the cell transfer, the growth medium is
replaced by phenol-free DMEM medium, thereby lowering the autofluorescence in-
tensity. Cells are transfected using Lipofectamine in Opti-MEM according to the
manufacturer's protocol (Invitrogen). Since the H1 DNA constructs used here con-
tain a CMV high expression promoter and FFS requires low concentration levels, the
amount of DNA used in the transfection procedure is only 1 ng per construct. The
FFS experiments are carried out half a day after transfection.
60% (
5.0
