viscous solutions such as Matrigel and collagen, leave the tip
immersed in the liquid for few seconds after releasing the
piston of the pipette allowing the proper aspiration of the
desired volume. Moreover, refrain from pushing down the
plunger after the first stop when dispensing the viscous fluids
to avoid the formation of air bubbles. Table 2 describes
volumes adjusted for a 9.5 mg/mL collagen I and 8.9 mg/
mL Matrigel stock solutions, respectively for a total of 1 mL gel
volume (see Notes 7 and 8).
2. With a 1 mL pipette, mix thoroughly avoiding bubble formation. Keep mixing slowly until the color of the solution
becomes salmon pink. Always work on ice to avoid premature
gel polymerization. Keep the solution on ice during the spheroid collection step.
3. Spheroid collection: tilt the lid and slowly flush 5 ml of culture
medium with a 5 mL pipette to concentrate the spheroids in
Fig. 2 Embedding the spheroids in the ECM gel mixture. (a) Spheroids are collected into a 15 mL tube by
washing out the drops with 5 mL of cell culture medium. (b) After the spheroids pellet, the culture medium is
removed. The tube is placed on ice and the gel mixture is added. (c) A 40 μL drop of ECM containing the
spheroids is deposited in each well of a 12-well glass-bottom plate. (d) The plate is inverted upside-down
every 2 min maintaining the plate at 37
C, 5% CO 2 . This step is repeated at least ten times
3D Invasion Assay with CAFs and Cancer Cells
249
immersed in the liquid for few seconds after releasing the
piston of the pipette allowing the proper aspiration of the
desired volume. Moreover, refrain from pushing down the
plunger after the first stop when dispensing the viscous fluids
to avoid the formation of air bubbles. Table 2 describes
volumes adjusted for a 9.5 mg/mL collagen I and 8.9 mg/
mL Matrigel stock solutions, respectively for a total of 1 mL gel
volume (see Notes 7 and 8).
2. With a 1 mL pipette, mix thoroughly avoiding bubble formation. Keep mixing slowly until the color of the solution
becomes salmon pink. Always work on ice to avoid premature
gel polymerization. Keep the solution on ice during the spheroid collection step.
3. Spheroid collection: tilt the lid and slowly flush 5 ml of culture
medium with a 5 mL pipette to concentrate the spheroids in
Fig. 2 Embedding the spheroids in the ECM gel mixture. (a) Spheroids are collected into a 15 mL tube by
washing out the drops with 5 mL of cell culture medium. (b) After the spheroids pellet, the culture medium is
removed. The tube is placed on ice and the gel mixture is added. (c) A 40 μL drop of ECM containing the
spheroids is deposited in each well of a 12-well glass-bottom plate. (d) The plate is inverted upside-down
every 2 min maintaining the plate at 37
C, 5% CO 2 . This step is repeated at least ten times
3D Invasion Assay with CAFs and Cancer Cells
249