the lower part of the lid. Transfer the spheroid suspension into
a 15 mL tube. Repeat this step to retrieve all the spheroids
(Fig. 2).
4. Wait a few minutes until all the spheroids pellet. Remove the
cell medium carefully without reaching the pellet and leave it
covered with 50 μL of medium. This step can be performed at
room temperature.
5. Transfer the tube to ice and let it cool down for 2 min. Calculate the required gel mixture volume to reach a concentration
of approximately 1 spheroid/10 μL (e.g., for 50 spheroids add
500 μL of gel mixture) (Fig. 2).
6. Mix well until all the spheroids are homogeneously dispersed in
the gel mixture.
7. On a 12-well glass-bottom plate, quickly deposit 40 μL drops
in each well. Refrain from pushing down the plunger after the
first stop while dispensing the matrix drops to avoid bubble
formation. Slowly turn the plate upside-down and incubate for
2 min at 37
C (see Note 11) (Fig. 2).
8. Slowly invert the plate right-side-up and incubate for two more
minutes.
9. Repeat the last two steps for 10–12 times. After about 20 min
the drops should become opaque, a sign of gel polymerization.
10. Incubate the plate 20 min at 37
C and 5% CO 2 while keeping
it upside-down (see Note 12).
11. Add 1 mL of culture medium in each well dispensing it slowly
on the border of the well to avoid detachment of the gel drops.
12. At this step, proper 3D embedding can be checked using a
standard inverted phase contrast microscope. The optimal
position of the spheroid is in the center of the drop, as spheroids located too close to the upper surface or to the glass
bottom may migrate in 2D. Incubate at 37
C and 5% CO 2
for 48–72 h until invasive strands are formed (incubation time
can vary depending on the cell type).
3.3 Image
Acquisition
Microscopy acquisition is performed at 37
C in a CO 2 humiditycontrolled chamber. Acquisition can be performed either semiautomatically or sequentially, recording a z-stack of each spheroid
separately. Here we describe a semi-automated procedure where
all positions are set prior to acquisition. This option can also be
used to run time-lapse imaging.
1. Insert 10Â objectives (e.g., Plan Fluor, NA 0.30, Ph1) (see
Note 9).
2. Carefully fix the plate inside the plate holder and cover with the
CO 2 chamber.
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Sefora Conti et al.
a 15 mL tube. Repeat this step to retrieve all the spheroids
(Fig. 2).
4. Wait a few minutes until all the spheroids pellet. Remove the
cell medium carefully without reaching the pellet and leave it
covered with 50 μL of medium. This step can be performed at
room temperature.
5. Transfer the tube to ice and let it cool down for 2 min. Calculate the required gel mixture volume to reach a concentration
of approximately 1 spheroid/10 μL (e.g., for 50 spheroids add
500 μL of gel mixture) (Fig. 2).
6. Mix well until all the spheroids are homogeneously dispersed in
the gel mixture.
7. On a 12-well glass-bottom plate, quickly deposit 40 μL drops
in each well. Refrain from pushing down the plunger after the
first stop while dispensing the matrix drops to avoid bubble
formation. Slowly turn the plate upside-down and incubate for
2 min at 37
C (see Note 11) (Fig. 2).
8. Slowly invert the plate right-side-up and incubate for two more
minutes.
9. Repeat the last two steps for 10–12 times. After about 20 min
the drops should become opaque, a sign of gel polymerization.
10. Incubate the plate 20 min at 37
C and 5% CO 2 while keeping
it upside-down (see Note 12).
11. Add 1 mL of culture medium in each well dispensing it slowly
on the border of the well to avoid detachment of the gel drops.
12. At this step, proper 3D embedding can be checked using a
standard inverted phase contrast microscope. The optimal
position of the spheroid is in the center of the drop, as spheroids located too close to the upper surface or to the glass
bottom may migrate in 2D. Incubate at 37
C and 5% CO 2
for 48–72 h until invasive strands are formed (incubation time
can vary depending on the cell type).
3.3 Image
Acquisition
Microscopy acquisition is performed at 37
C in a CO 2 humiditycontrolled chamber. Acquisition can be performed either semiautomatically or sequentially, recording a z-stack of each spheroid
separately. Here we describe a semi-automated procedure where
all positions are set prior to acquisition. This option can also be
used to run time-lapse imaging.
1. Insert 10Â objectives (e.g., Plan Fluor, NA 0.30, Ph1) (see
Note 9).
2. Carefully fix the plate inside the plate holder and cover with the
CO 2 chamber.
250
Sefora Conti et al.