2. Software to program multiple positions, z-stack and multiple
wave lengths acquisition.
3. 10Â objective (e.g., Plan Fluor, NA 0.30, Ph1) (see Note 9).
4. CO 2 chamber and temperature control (set at 37
C).
5. Image analysis software (e.g., ImageJ/FIJI, Imaris or
equivalent).
3 Methods
3.1 Hanging-Drop
Method for Spheroid
Self-Assembly
1. Trypsinize HCC1806 cells and CAFs from a 60% confluent
flask to single-cell suspension. Remove the cell culture
medium, wash with PBS and add 1 mL of Trypsin EDTA 1Â
for a 75 cm
2 flask or 0.5 mL for a 25 cm
2 flask. Incubate at
37
C until the cells detach (approximately 2 min incubation).
Neutralize the trypsin by adding 10 mL culture medium and
transfer to a 15 mL centrifuge tube. Mix well and keep 10 μL
for counting cells (can be added directly to hemocytometer
chamber).
2. Pellet cells by centrifugation. Aspirate the supernatant and
resuspend the cell pellet with 3 mL of culture medium containing 2 μL of CellTracker staining solution (10 mM). Incubate
each cell type with the chosen CellTracker Dye for 30 min at
37
C (see Note 4).
3. Count HCC1806 cells and CAFs. Determine the resuspension
volume needed to reach a concentration of 750,000 cells/mL.
4. After incubating for 30 min, dilute the CellTracker staining
solution with the appropriate culture medium for each cell type
up to 15 mL.
5. Centrifuge both tubes containing HCC1806 cells and CAFs
and aspirate the supernatant.
6. Resuspend the cells with previously calculated volumes.
7. In a 1.5 mL centrifuge tube, prepare the cell suspension solution for spheroid preparation (see Table 1). The detailed
volumes refer to 1:1 and 2:1:1 ratio between CAFs and cancer
cells respectively. These proportions and the number of different cell types added may be adjusted according to the requirements of each experiment.
8. Fill the bottom of a sterile Petri dish with 10 mL sterile PBS to
create a humid chamber (PBS will prevent the suspended drops
from drying during spheroid formation) (Fig. 1).
9. Mix thoroughly the cell suspension solution and plate 20 μL
drops on the lid of the Petri dish. Avoid bubble formation
3D Invasion Assay with CAFs and Cancer Cells
247
wave lengths acquisition.
3. 10Â objective (e.g., Plan Fluor, NA 0.30, Ph1) (see Note 9).
4. CO 2 chamber and temperature control (set at 37
C).
5. Image analysis software (e.g., ImageJ/FIJI, Imaris or
equivalent).
3 Methods
3.1 Hanging-Drop
Method for Spheroid
Self-Assembly
1. Trypsinize HCC1806 cells and CAFs from a 60% confluent
flask to single-cell suspension. Remove the cell culture
medium, wash with PBS and add 1 mL of Trypsin EDTA 1Â
for a 75 cm
2 flask or 0.5 mL for a 25 cm
2 flask. Incubate at
37
C until the cells detach (approximately 2 min incubation).
Neutralize the trypsin by adding 10 mL culture medium and
transfer to a 15 mL centrifuge tube. Mix well and keep 10 μL
for counting cells (can be added directly to hemocytometer
chamber).
2. Pellet cells by centrifugation. Aspirate the supernatant and
resuspend the cell pellet with 3 mL of culture medium containing 2 μL of CellTracker staining solution (10 mM). Incubate
each cell type with the chosen CellTracker Dye for 30 min at
37
C (see Note 4).
3. Count HCC1806 cells and CAFs. Determine the resuspension
volume needed to reach a concentration of 750,000 cells/mL.
4. After incubating for 30 min, dilute the CellTracker staining
solution with the appropriate culture medium for each cell type
up to 15 mL.
5. Centrifuge both tubes containing HCC1806 cells and CAFs
and aspirate the supernatant.
6. Resuspend the cells with previously calculated volumes.
7. In a 1.5 mL centrifuge tube, prepare the cell suspension solution for spheroid preparation (see Table 1). The detailed
volumes refer to 1:1 and 2:1:1 ratio between CAFs and cancer
cells respectively. These proportions and the number of different cell types added may be adjusted according to the requirements of each experiment.
8. Fill the bottom of a sterile Petri dish with 10 mL sterile PBS to
create a humid chamber (PBS will prevent the suspended drops
from drying during spheroid formation) (Fig. 1).
9. Mix thoroughly the cell suspension solution and plate 20 μL
drops on the lid of the Petri dish. Avoid bubble formation
3D Invasion Assay with CAFs and Cancer Cells
247