Chapter 2 – Materials and Methods
2.6 Confocal microscopy
HEPES-buffered saline solution (HBSS; 147 mM NaCl, 24 mM KCl, 1.3 mM
CaCl2, 1 mM MgSO4, 10 mM HEPES, 2 mM sodium pyruvate, 1.43 mM NaHCO3,
pH 7.45) containing 4.5 mM D-glucose was used as the imaging buffer in all
experiments outlined below and was pre-warmed before use to the stated
temperature in which the experiments were carried out. Imaging buffer was
used to dilute both BODIPY-TMR-CGP and BODIPY630/650-S-PEG(8)-
propranolol fluorescent ligands from a 1 mM DMSO stock to achieve the
DMSO
0.01 % (v/v; final concentrations), reducing potential harmful effects of this
solvent on the cells (Brayton, 1986). Incubation of cells in a cell culture
incubator refers to a 5 % CO2/95 % air atmosphere.
SNAP-tag labelling
C SNAP D A 1-adrenoceptors
were grown to confluence in 8-well plates as described above (see Cell
culture). Prior to experimentation, the growth medium was removed off the
cells and replaced with fresh medium containing 1 µM of the cell
impermeable SNAP-tag substrate SNAP-Surface® 488 (BG-488; New England
Biolabs, Ipswich, MA). The cells were then incubated at 37 °C in a cell culture
the medium containing the SNAP-tag substrate was removed and the cells
were washed twice in imaging buffer (pre-warmed to 37 °C), before
incubating the cells in 200 µL imaging buffer in a cell culture incubator (37 °C)
for a further 30 minutes in the dark. Finally, the imaging buffer was replaced
with 400 µL fresh imaging buffer and the cells used immediately for imaging.
Saturation binding experiments
The protocol used here was based on that described by Baker et al. (2003d)
BODIPY TMR CGP CHO 2-adrenoceptor.
BODIPY-TMR-CGP and BODIPY630/650-S-PEG8-propranolol saturation binding
experiments were performed at room temperature (circa 21 °C) using CHO-
1 CS CHO 2-CS cells seeded into 8-well plates (as described in Cell
culture). Immediately prior to experimentation, the growth medium was
removed and the cells were then washed once with imaging buffer, before
the addition of 360 µL imaging buffer to each well. The 8-well plate was
placed onto the microscope stage and 40 µL (i.e. 10x dilution to final
concentration) of a range of concentrations from 3-100 nM (final
concentrations) of either BODIPY-TMR-CGP or BODIPY630/650-S-PEG8-
propranolol were added to designated wells. The cells were exposed to the
fluorescent ligand for 10 minutes before imaging. To ensure strict exposure
time for all cells and to allow circa 5 minutes to image each well, the addition
of fluorescent ligands to designated wells was staggered.
This method was also based on that described by Baker et al. (2003d) for the
BODIPY TMR CGP CHO 2-adrenoceptors. BODIPY-
TMR-CGP and BODIPY630/650-S-PEG(8)-propranolol inhibition binding
experiments were performed at room temperature (circa 21 °C) using CHO-
1 CS CHO 2-CS cells seeded into 8-well plates (as described in Cell
culture). CHO-CS cells were used as a negative control. On the day of
experimentation, the growth medium was removed and the cells washed
once with pre-warmed imaging buffer, before 360 µL of imaging buffer was
added to the positive control well, measuring total binding, and the negative
control well (containing CHO-CS cells), measuring non-specific binding levels.
360 µL of imaging buffer containing increasing concentrations of the
competitor (0.01-1000 nM) was added to the designated wells. The cells were
then incubated for 30 minutes at 37 °C in a cell culture incubator. Following
this, the plate was mounted on the microscope stage and the experiment
started by the addition of 40 µL of the desired concentration of the
fluorescent ligand to the first well, which was imaged after a further 10
minutes. To ensure the same exposure time of cells to the fluorescent ligand
in all wells and to allow a 5 minute imaging time for each well, the addition of
the fluorescent ligand and imaging of the wells was staggered and carefully
timed. Where cells expressing SNAP-tagged receptors were used, the SNAP-
tag labelling was performed first (as described above) and the 30 minute
incubation step following the washout of the SNAP-tag substrate was
Internalisation experiments
F SNAP 1-adrenoceptor cells were
seeded into 8-well plates as described in Cell culture and the SNAP-tagged
receptors were labelled as described above, but using 360 µL imaging buffer
in the final incubation step. Next, the 8-well plate was mounted onto the
microscope stage. The unlabelled ligands to be used were diluted in imaging
buffer to the desired concentrations (10x final concentration). Before addition
of any ligands, the first well was imaged to allow optimisation of microscope
settings (laser power, gain and offset), which were then kept constant for all
other wells throughout the experiment. Following this, 40 µL of the ligand
was added to the first well and imaged immediately (at a different site than
that used to determine the microscope settings; time point 0 minutes). After
60 minutes incubation of the ligand, the well was imaged again (time point 60
minutes).
Bimolecular fluorescence complementation (BiFC) experiments
Using the bimolecular fluorescence complementation (BiFC) approach, two
non-fluorescent N- and C-terminal halves of a fluorescence protein (e.g.
yellow fluorescence protein, YFP) are attached to two potentially interacting
proteins (e.g. dimerising receptors). Upon receptor dimerisation, the two non-
fluorescent YFP halves then come together and reconstitute the full length
fluorescent YFP, thus the detection of YFP fluorescence indicates an
interaction of the two proteins studied and was used in this thesis to detect
seeded into wells of an 8-well plate (day 1) and transiently transfected with
YFPN-tagged and YFPC 1-adrenoceptor recombinant DNA the
following day (day 2) as described in Generation of new cell lines. The next
day (day 3), the transfection medium was removed off the cells and replaced
with fresh growth medium, before the cells were placed back into the cell
culture incubator (37 °C, 5 % CO2/95 % air atmosphere). After circa 6 hours,
the cells were then moved into a 30 °C incubator (5 % CO2/95 % air
atmosphere) to allow the maturation of the fluorophore following correct
protein folding. On day 4, the cells were used for experimentation.
Confocal imaging
All confocal imaging experiments were performed on the Zeiss LSM710
confocal microscope (unless otherwise stated) with a Zeiss 40x1.3NA oil
immersion lens (Zeiss, Jena, Germany). The laser that emits light at a
wavelength closest to the wavelength needed to cause maximum excitation
of the chosen fluorophore was used. Upon excitation, the fluorophore then
emits light itself at a longer wavelength, which is captured using the
appropriate wavelength filter. The fluorophores, lasers and microscope
settings used throughout this thesis are summarized in Table 2.2. The pinhole
diameter used in all confocal experiments was 1 airy unit (AU), which
represents a near optimal setting that reduces out-of-focus emission
contributions without the loss of intensity of the measured fluorophore. The
pinhole setting and the laser wavelength used determine the optical slice
the thinner the optical slice). The first well of an 8-well plate was used for
calibration in each experiment. The cells were imaged and the range indicator
was set to determine brightness (detector gain) of the bound ligand and the
contrast (amplitude offset) such that pixels were either over-saturated or
below the detection limit. The maximal image brightness was then set to a
value greater than that achieved with the control well so that brighter binding
could be detected. The confocal settings (laser power, digital values for image
brightness, background and contrast) were then kept constant for the rest of
the experimental day. This allowed direct comparisons between wells to be
made. This area of the first well used to determine the settings was subjected
to laser exposure much more than any other area and hence had potential for
more photobleaching. A second area of the well was therefore selected for
Table 2.2 Excitation and emission wavelengths of the fluorescent molecules used in studies within this thesis are listed together with the lasers and microscope settings used to image cells treated with these fluorescent molecules.
fluorophore excitation/emission laser type laser wavelength
Microscope settings used
emission filter gain offset optical slice
BG-488 506/526 Argon 488 nm 505 nm long-pass 600-1100 0-1 0.37 µm
BODIPY-TMR-CGP 545/570 Helium-Neon 543 nm 550 nm long-pass 600-1100 0-1 0.40 µm
BODIPY-TMR-CGP 545/570 Helium-Neon 561 nm 565 nm long-pass 600-1100 0-1 0.41 µm
BODIPY630/650-S-PEG(8)-propranolol 630/650 Helium-Neon 633 nm 650 nm long-pass 600-1100 0-1 0.46 µm
Where two lasers were used at the same time (e.g. 488 nm and 561 nm lasers
BODIPY TMR CGP SNAP 1-adrenoceptors), the
multitracking facility in the Zeiss imaging software was used. This allowed the
sample to be illuminated with one laser at a time in order to avoid any bleed
through of the laser used (e.g. 488 nm) causing excitation of the fluorophore
used (e.g. BODIPY-TMR-CGP). For the same reason, the emission filter of the
higher energy fluorophore was also adjusted (e.g. 505-550 nm band-pass
filter was used to capture BG-488 fluorescence). Furthermore, the pinhole
(thus the optical slice thickness) was set for the highest wavelength laser used
(i.e. 561 nm laser, if both 488 and 561 nm laser where used) and the optical
slice thickness was then matched for the second laser. Using two lasers also
increased the total well laser exposure time. To correct for that, fewer frames
were taken (4-8 frame scans, 1024x1024 pixels), thus limiting each image to
approximately one minute laser exposure.
Data collection
Using 8-well plates limits the number of experimental conditions that can be
tested in a given experiment. In the experiments described above, every well
represented a different experimental condition (e.g. different concentration
of fluorescent ligand or inhibitor ligand or internalising ligand). However, 2-4
different areas were imaged within a given well and each image was analysed
as described below, thus providing duplicate to quadruplicate measures per
thesis for these experiments, however, refer to number of separate
experiments set up and carried out (i.e. different experimental days).
Total image intensity analysis
All images taken on the Zeiss LSM710 microscope were captured using an 8bit
greyscale, which allows for 256 different intensity levels of a given pixel (from
0-255; where 0 represents the weakest and 255 the strongest fluorescence
intensity). All data analysis was carried out using Zeiss Zen2010 software (Carl
Zeiss, Jena, Germany). This provides the frequency of pixels recorded at each
of the 256 greyscale intensities for each image taken. The total image
intensity (arbitrary units) is calculated as the sum of the product of frequency
x greyscale intensity for each of the 256 grey scale intensity values. This value
was then divided by the total number of pixels per image (1024x1024 for the
total image) to give the average pixel intensity.
Region of interest (ROI) analysis
Where stated, regions of interest were drawn around the membrane of a
given number of cells (stated for each experiment) in each image taken. The
Zeiss software provides the frequency of pixels recorded at the 256 greyscale
intensities and the number of pixels in the combined area of the drawn
regions of interest of one image, and thus allows quantitative analysis as
described above using average pixel intensities that refer to the regions of
Co-localisation analysis
Where two different wavelengths were used to image two different
fluorescently labelled molecules (e.g. SNAP-tagged 1-adrenoceptor using 488
nm and BODIPY-TMR-CGP binding using 561 nm excitation wavelengths), the
individual images obtained for each wavelength were merged. These images
are shown in colour, where yellow pixels clearly identify regions of spatial
overlap of the two fluorescently labelled molecules. The fluorescence
intensities of each individual channel, however, are shown in monochrome to
allow better visualisation of the greyscale intensities of the pixels in each
channel (from 0, i.e. black, to 255, i.e. white pixels).
Where used, a co-localisation plot was obtained and a crosshair placed on this
plot to highlight the region of co-localised pixels (region 3). To do this, a
region of interest was drawn on the background of the image away from the
cells and the average intensity and its standard deviation (SD) of this region
were collected in each of the two channels. The crosshair was then placed to
the intensity levels calculated from adding the average background pixel
intensity to 2x its standard deviation. This represents the background
fluorescent intensity. Pixels with intensity values greater than those
calculated in both channels will be located in region 3 and represent co-