next generation wound healing assay | application note · throughout the time-lapse sequence. d) t...
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Go beyond traditional wound healing assays with the Phasefocus Livecyte
• Directly measure cell motility
• Separate cell motility from cell proliferation
• Segment and track individual cells along the leading edge
• Characterise morphological and behavioural cell phenotypes during wound healing
Next Generation Wound Healing Assay | Application Note
Introduction
The Livecyte system can be used to image the
cells and analyse their behaviour as both an overall
population and as single individual cells. The non-
invasive nature of the measurement (label-free,
low illumination power) ensures that the cells
are unperturbed and behave in a more natural
manner over an extended period of time. Very
large field of views are available without stitching,
which allows for statistically significant population
metrics to be extracted (gap closure, proliferation),
while the quantitative, high contrast nature of
the images allows for single cell behaviour to be
characterised (morphological parameters and
motility). The Livecyte system generates a far
greater suite of information from such established
assays, compared to traditional methods such
as brightfield, fluorescence or phase contrast.
Methods
MDA MB231 cells were seeded at 5x105 cells/ml
in Ibidi culture-insert 2 well dishes (Cat. 81176) to
create a cell free gap by exclusion. 24 hours after
seeding the insert was removed and the cells were
treated either with a vehicle control (1% BSA\HBSS)
or with 50μM Lysophosphatidic acid (LPA). The
Phasefocus Livecyte system was used to perform
time-lapse imaging of the wound, every 10 minutes
for a total period of 48 hours. The Phasefocus
CAT (Cell Analysis Toolbox) software was used to
segment and measure wound area, from which
t1/2 gap (time for area of wound to reduce by half)
and Vmigration (cell migration rate) was calculated.
In addition, individual cells were segmented and
tracked to report motility and morphological data.
The wound healing assay is an established in vitro procedure that measures collective cell migration. The basis of this assay is to create a cell free region within a confluent layer of cells, either through the creation of a scratch by removing the cells post adherence, or through physical exclusion of cells during seeding. Once the cell free area has been created the closure of the wound is monitored through live-cell imaging. The measure of collective cell migration in this manner, replicates sheet migration that occurs in a range of biological processes such as; cancer metastasis, embryonic morphogenesis and tissue injury.
Figure 1a).
Figure 1d). Figure 1e).
Figure 1 - Measuring Gap closure using the Phasefocus CAT software a) Representative images of wound gap segmented using the CAT software (blue outline), at 0, 24 and 48 hours and for both control and LPA treated cells. b) Relative gap area and c) relative dry mass measured throughout the time-lapse sequence. d) t1/2 gap closure and e) collective migration calculated from the GAP closure.
(Time hours)12 18 24 30 36 42 48
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Wound Area (control) Wound Area (50µM LPA)
(Time hours)12 18 24 30 36 42 48
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Figure 1b). Figure 1c).
0 Hours 24 Hours 48 Hours
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Results
The rate of gap closure is the most common metric that
is measured from a wound healing assay. Subsequently,
the t1/2 of the gap and Vmigration can be calculated. The
Livecyte system was utilised to accurately determine
the change in gap area over a 48-hour period (Fig.
1a). The addition of 50μM LPA caused the wound to
close at a faster rate (Fig. 1a, b) with a t1/2 gap of 21.4
hours and Vmigration of 5.3μM/hour when compared
to the control (41.9 hours and 2.5μM/hour, respectively.
Fig. 1 d, e.) However, the change in gap area can be
influenced by both cell migration and cell proliferation.
It is therefore important to account for any changes
induced as a result of cell proliferation. The quantitative
nature of images acquired allows for the determination
of optical volume of cells. Measuring total optical
volume of all cells over time, gives an indication of the
rate of cell growth and proliferation. In this experiment
cells treated with 50μM LPA increased the rate of cell
proliferation after 16 hours (Fig 1c). This indicates that
post 16 hours both cell migration and proliferation had
an influence on t1/2 gap and Vmigration. In addition to
modelling collective migration, the additional capabilities
of the Livecyte system allow for direct measurement
of single cells at the leading edge of the wound (Fig.
2a). Tracking such cells revealed an increase in mean
cell speed and instantaneous cell speed following
application of 50μM LPA. The LPA treatment also
induced a reduction in the meandering index, revealing
that the behaviour of migration was also influenced
(Fig. 2b). Further to motility parameters the virtue of
individual cell segmentation, allowed for quantification
of morphological parameters such as cell area,
thickness, sphericity and length/width ratio. Treatment
with 50μM LPA caused an increase in cell thickness
and sphericity, whilst reducing the length/width ratio.
Conclusion
In this application note we have demonstrated the advanced capabilities of the Phasefocus Livecyte system to image and analyse the wound healing assay. The non-invasive nature of the measurement allows for higher temporal intervals to be imaged and thus a more accurate measure of wound closure over time to be achieved. Further to information regarding collective migration, the high contrast, quantitative images produced, enable multi parametric analysis to be performed at a single cell level. This single-cell analysis directly measures cell migration, while the morphological information extracted, provides both a behavioural and morphological analysis of individual cells during the wound healing process.
Figure 2 - Advanced segmentation and tracking of single cells at the leading edge a) Representative images of cells at the leading edge of the wound segmented and tracked using the Phasefocus CAT software over 48 hours. b) Motility and c) Mean morphological metrics calculated from the tracked cells.
Cel
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Figure 2a).
Figure 2b). Figure 2c).
0 Hours 24 Hours 48 Hours
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T: +44 (0)114 286 6377
Phase Focus Limited
Electric Works
Sheffield Digital Campus
Sheffield
S1 2BJ
UK
www.phasefocus.com
For more information on the benefits of the
Livecyte system, to access application notes
and for additional product information, please
visit: www.phasefocus.com/livecyte
A sample of time-lapse videos can be found at:
www.youtube.com/phasefocuslimited
© Phase Focus Limited. AN 001 | May 2016