A B EGFP EGFP
3.2.9 Preparation o f coverslips
18 mm X 18 mm, thickness no. 1.5 glass coverslips were prepared for use in random walk,
chemotaxis, and fixed-cell FRET/FLIM assays. Coverslip dimensions were primarily chosen
for their compatibility with the Dunn chemotaxis chamber and to enable the optimal
microscopic imaging o f cells at both low and high magnification (the majority o f modem
microscope objectives are optimised for use with 170 pm thick (no. 1.5) glass coverslips).
Coverslip cleaning: Coverslips were thoroughly washed prior to tissue culture use: coverslips were placed in Teflon coverslip racks (molecular probes) and immersed in a mixture o f 60 %
concentrated HCl, 40 % ethanol (95 % pure) for 2 hrs. Racks were subsequently removed
from the acid/alcohol mixture and immersed in ddHzO for three sequential 10 min washes.
Coverslips were then stored under 70 % ethanol until required. Immediately before use
coverslips were dried mechanically using an air hose with fitted ployVENT™ 200 nm pore
filter (Whatman). Passive evaporation was not permitted as a method o f drying as this often
resulted in the formation o f residue on the coverslip surface.
Coverslip etching: In cases where cell cultures were prepared for microinjection and subsequent analysis in chemotaxis experiments, coverslips were marked to facilitate the
relocation o f the microinjected cells. When correctly assembled the Dunn chemotaxis
chamber permits the observation of cells within only a small central region o f the coverslip
approximately 2 mm x 2 mm in area. To ensure microinjected cells could be observed within
the chamber the central region o f each coverslip was marked with a ‘window’ pattern, and
cells only microinjected within this area. A manually controlled xy stage with graduated axes and a fitted diamond objective was used to accurately etch the window pattern into the centre
o f each coverslip.
3,2,10 Application o f the Dunn chemotaxis chamber
Preperation o f subconfluent cultures: Cells were plated on washed, pre-etched coverslips placed in 35 mm sterile plastic tissue culture dishes (Coming) containing 3 ml o f the
appropriate culture medium for the cell type used. Freshly trypsonised X I5 rat sarcoma cells,
NIH 3T3, and Swiss 3T3 fibroblasts were plated at 5 x 10^, 2 x 10^ and 1 x 10^ cells per dish
reverse surface to that o f the scratched markings: many cell types are known to exhibit
contact guidance in vitro, preferentially aligning and migrating along linear surface markings (Stepien et al., 1999). Since chemotaxis was the subject o f investigation it was essential to
minimize the influence o f other environmental factors capable o f influencing the direction of
cell migration. Dishes were briefly shaken in a horizontal figure-of-eight pattern to facilitate
the even spreading o f cells before returning to the incubator.
Nuclear microinjection: T15 rat sarcoma cells were transferred to serum reduced (0.5 % BS), phenol red free culture medium three hours prior to microinjection and six hours prior to
Dunn chamber assembly. Swiss fibroblasts were transferred to full serum (10 % PCS),
phenol red free culture medium supplemented with 20 mM HEPES, pH 7.5 prior to
microinjection to compensate for the usual CO2 requirements.
Cells positioned within the central etched region o f a coverslip were located on the
microscope by focusing on the lower coverslip surface, detecting the etches, and then
refocusing through the coverslip onto the cells. A group o f evenly spaced cells within the
etched region was selected and approximately every other cell microinjected in the nucleus.
Expression constructs encoding EGFP fusions o f proteins of interest were all microinjected at
a concentration o f 0.05 pg/pl. Cells were allowed 3 hours to express microinjected constructs
before coverslips were assembled onto Dunn chemotaxis chambers. For detailed description
Preparation o f growth factors: Lyophilized recombinant rat PDGF-BB (Sigma) and recombinant human IGF-1 (PeproTech) were reconstituted in ddHzO adjusted to pH 7.0 and
supplemented with 0.1 mg/ml BSA (Sigma) carrier protein to a concentration of 50 pg/pl and
stored as separate 20 pi stock aliquots at -2 0 °C until required.
Aliquots containing culture medium with and without growth factors were prepared for Dunn
chamber assembly. For T15 rat sarcoma cells reduced serum (0.5 % BS), phenol red free
culture medium was used throughout. Immediately prior to chamber assembly growth factors
were added to culture medium to a final concentration o f 60 ng/ml PDGF-BB, 80 ng/ml
IGF-1. For Swiss and NIH 3T3 fibroblasts full serum (10 % FCS), phenol red free medium
supplemented with 20 mM HEPES adjusted to pH 7.5 was used throughout. PDGF-BB alone
provided the chemotactic stimulus for Swiss and NIH 3T3 fibroblasts and was added to
culture medium to a final concentration of 100 ng/ml.
Chamber assembly: Assembly o f the Dunn chemotaxis chamber (Weber Scientific International) is summarized in Figure 37, page 19, and is described in detail here. The outer
and inner wells o f the chemotaxis chamber were flooded with culture medium before
inverting the glass coverslip with adherent cells carefully onto the chamber using forceps.
Care was taken to prevent bubbles from forming between the underside o f the coverslip and
the inner and outer wells. The volume o f culture medium was sufficient to allow the
coverslip to float above the surface o f the chamber enabling it to be repositioned with the
fingertips without crushing the cells. In cases where microinjected cells were present, a
Figure 37. Assembly o f the Dunn chemotaxis chamber
Procedure fo r assembling the Dunn chemotaxis chamber. (A) The central region o f the chamber was flooded with culture medium. (B) The coverslip with adherent cells was then inverted and lowered onto the chamber. The coverslip was positioned such that a small filling slit was left at one end o f the outer well (arrow). Tissue paper was used to carefully dry the edges o f the coverslip so that it lay flush against the surface o f the chamber. (C) The coverslip was then sealed to the chamber using the hot wax mixture. At this stage the edge that form ed the filling slit was not sealed. (D) Tissue paper was used to draw medium out from the outer well. (E) The outer well was washed once with fresh medium and the chemoattractant then applied using a pipette. (F) The edge o f the filling slit was dried and finally sealed with hot wax mixture.
position them over the annular platform, roughly parallel to the outer well. If the fluorescent
cells had been microinjected within the central 2 mm x 2 mm etched area o f the coverslip
then the coverslip would be positioned such that a thin section o f the outer well remained
exposed at the opposite end of the chamber. This exposed region formed the ‘filling slit’ that
enabled access to the outer well for the addition o f culture medium containing the
chemoattractant. The sides o f the coverslip were gently dried using sterile tissue paper,
taking care not to press down on the region suspended over the inner and outer wells as this
could easily result in crushing the cells. Furthermore it was essential to avoid moving the
coverslip after the fluorescent cells had been positioned over the annular platform. Once the
coverslip perimeter was dry it was sealed to the chamber using a hot wax sealing mixture
consisting o f a 1:1:1 ratio o f paraffin wax (Fischer Scientific), beeswax (Acros Organics),
and petroleum jelly (Fischer Scientific) mixed and heated to 60 °C. The sealing mixture was
applied using a paintbrush. The coverslip edge that formed the filling slit was not sealed at
this stage. Medium within the outer well o f the chemotaxis chamber was drawn out by
applying sterile tissue paper to the edge o f the filling slit. Suction pressure prevented the
removal o f medium from within the diffusion gap and the central well. The outer well was
then washed once by the re-addition of fresh culture medium using an Eppendorf pipette.
This was done to remove any factors released from crushed cells at the chamber perimeter
that could have potentially influenced the behaviour of cells under observation. The fresh
medium was then removed with tissue paper in the same way, and the culture medium
containing the chemoattractant then applied. The; edge o f the filling slit was then carefully
Recording o f cell behaviour: Digital time-lapse microscopy was used to record cell behaviour in all Dunn chamber chemotaxis experiments. Cells were imaged on either a Zeiss
Axiovert TV 135 (Carl Zeiss) or a Nikon Diaphot (Nikon) inverted microscope. The
specifications o f the Zeiss microscope are described in: time-lapse microscopy 2.2.3,
page 19. As with the Zeiss system the Nikon microscope was optimised for the multi-channel
time-lapse recording o f live mammalian cells in culture. The microscope was fitted with
tungsten and mercury lamps, separate bright field and fluorescence shutters (Uniblitz), a
shutter controller (Ludle), and a sensitive CCD camera (Orcar ER, Hamamatsu). Peripheral
devices were under the control of acquisition manager software. A filter block containing
appropriate excitation and emission filters and a dichromatic mirror enabled the visualization
o f EGFP fluorescence (Omega, FX100-2 Alpha Vivid). The microscope was positioned on
an optical table isolator and housed in a Perspex environment chamber accurately maintained
at 37 °C.
The chemotaxis chamber was inverted and placed on the microscope in a conventional slide
holder and positioned so that the diffusion gap was directly above the objective. The image
acquisition software was then used to select a suitable observation field within the diffusion
gap that contained the majority o f the fluorescent cells. The innermost annular edge o f the
outer well was positioned at the top o f the computer screen by rotating the CCD camera, thus
aligning the chemotactic gradient with the vertical axis o f the image. The chamber was then
left to stand in the microscope for 20 min to allow the temperature to equilibrate and the
spaced cells above the diffusion gap and furthest from the filling slit was selected for
observation.
Low magnification multi-channel digital time-lapse microscopy was used to monitor cell
behaviour in the chemotactic gradient over a 16-hour period. Sequential phase contrast and
EGFP fluorescence images were acquired every 5 min using either a xlO NA 0.3 or x20 NA
0.5 phase contrast objective. A total o f 200 time points were acquired resulting in films of
1000 minutes duration (-16.7 h).