3. Turn on the microscopy software.
4. Using bright-field live imaging, find a spheroid and assess
optimal laser power and exposure time for each fluorescent
laser channel.
5. In the multi-acquisition tab, select z-stack option, relative z,
start with 0 and end with 200–250 μm with a step of 1–10 μm
depending on the desired resolution and the spheroid size. In
the described experimental setting, spheroid diameter varies
from 200 to 250 μm approximately, but this depends on the
cell type used. Set acquisition channels with the optimal exposure times established in step 4.
6. Set the position focus on the lowest z plane of the spheroid.
Stack acquisition will start from that plane, moving upward to
the uppermost plane of the spheroid. When running time-lapse
acquisition we used an automated focus system to avoid focus
drift. If your microscope is not equipped with such system, you
may need to manually adjust the focus at regular intervals
during acquisition and/or set-up wider z-stacks to avoid the
sample to go out of the field of view during the acquisition (see
Note 9).
7. Select destination folder for saving images.
8. Once all positions are set, run the experiment.
3.4 Image Analysis
Image analysis is performed using the open source ImageJ/FIJI
[12]. Open a stack in ImageJ and using the ImageJ z-projection
function, under the “Stacks” tab, process all stacks to obtain
maximum-intensity projections of the saved z-series (acquired
with the 10Â objective). From each z-stack, the number and the
length of invading strands as well as the identification of leader/
follower cells within each strand can be assessed as follows (Fig. 3)
(see Note 13):
1. Calibrate spatially your image setting appropriate image scale
based on the camera pixel-size value and magnification (in this
experiment, camera pixel-size value: 0.645 μm/pixel using a
10Â magnification objective).
2. Select the straight-line tool. Draw a line starting from the
strand base, at the spheroid circumference, to its tip. Measure
its length. Repeat this step for all strands, evaluating both
length and number of invading strands (Fig. 3a).
3. From time-lapse images, identify leader/follower dynamics
(Fig. 3b).
3D Invasion Assay with CAFs and Cancer Cells
251
4. Using bright-field live imaging, find a spheroid and assess
optimal laser power and exposure time for each fluorescent
laser channel.
5. In the multi-acquisition tab, select z-stack option, relative z,
start with 0 and end with 200–250 μm with a step of 1–10 μm
depending on the desired resolution and the spheroid size. In
the described experimental setting, spheroid diameter varies
from 200 to 250 μm approximately, but this depends on the
cell type used. Set acquisition channels with the optimal exposure times established in step 4.
6. Set the position focus on the lowest z plane of the spheroid.
Stack acquisition will start from that plane, moving upward to
the uppermost plane of the spheroid. When running time-lapse
acquisition we used an automated focus system to avoid focus
drift. If your microscope is not equipped with such system, you
may need to manually adjust the focus at regular intervals
during acquisition and/or set-up wider z-stacks to avoid the
sample to go out of the field of view during the acquisition (see
Note 9).
7. Select destination folder for saving images.
8. Once all positions are set, run the experiment.
3.4 Image Analysis
Image analysis is performed using the open source ImageJ/FIJI
[12]. Open a stack in ImageJ and using the ImageJ z-projection
function, under the “Stacks” tab, process all stacks to obtain
maximum-intensity projections of the saved z-series (acquired
with the 10Â objective). From each z-stack, the number and the
length of invading strands as well as the identification of leader/
follower cells within each strand can be assessed as follows (Fig. 3)
(see Note 13):
1. Calibrate spatially your image setting appropriate image scale
based on the camera pixel-size value and magnification (in this
experiment, camera pixel-size value: 0.645 μm/pixel using a
10Â magnification objective).
2. Select the straight-line tool. Draw a line starting from the
strand base, at the spheroid circumference, to its tip. Measure
its length. Repeat this step for all strands, evaluating both
length and number of invading strands (Fig. 3a).
3. From time-lapse images, identify leader/follower dynamics
(Fig. 3b).
3D Invasion Assay with CAFs and Cancer Cells
251