HCC1806 cells were grown in RPMI 1640 Medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL
streptomycin and 1% of MEM Nonessential Amino Acid Solution (100Â).
3. Trypsin EDTA solution 1Â.
4. Phosphate buffered saline sterile.
2.2 Hanging-Drop
Method for Spheroid
Self-Assembly
1. Sterile syringe filter with a 0.22 μm pore size hydrophilic Polyethersulfone (PES) membrane, 33 mm diameter.
2. 1.2% Methylcellulose solution: Weight 0.6 g Methylcellulose
and transfer into a 50 mL tube with a magnetic stirring bar.
Make up to 50 mL with serum-free DMEM high glucose. Stir
until fully dissolved (it might take several minutes). Filter with a
0.22 μm filter, aliquot and store at 4
C (see Note 2).
3. CAFs culture medium (see Note 3), warmed at 37
C
before use.
4. 0.5% Trypsin EDTA, warmed at 37
C before use.
5. PBS buffer, sterile.
6. Cell labeling: In order to visualize the different population of
cell in the invasion assay, several strategies of cell labeling by
fluorescent dyes can be used. In this protocol we use CellTracker™ Green CMFDA Dye and Deep Red Dye staining
solution (Invitrogen) to stain the CAFs and cancer cells respectively (see Note 4).
7. Sterile Petri dishes (100 mm).
8. 1.5 mL sterile centrifuge tubes.
9. Hemocytometer chamber for cell counting.
10. Cell mixture: see Table 1.
2.3 Embedding
Spheroids into Matrix
1. 5ÂCol solution (see Note 5): Weigh 2.5 g of DMEM powder,
high glucose, pyruvate, and 1 g Sodium Bicarbonate
(NaHCO 3 ) transfer into a 50 mL tube. Add 5 mL of 1 M
Hepes pH 7.5 and make up to 50 mL with milli-Q water. Stir
until the powder is completely dissolved. Filter with a 0.22 μm
filter and store at 4
C.
2. Rat tail collagen type I. Store at 4
C. The stock concentration
ranges between 8 and 11 mg/mL (see Note 6).
3. Matrigel matrix (see Note 7). Make aliquots and store at
À20
C. Avoid multiple freeze-thaws. Thaw overnight at
4
C in ice one day before use.
4. Fetal bovine serum (FBS), filtered with a 0.22 μm filter. Keep at
4
C until use.
3D Invasion Assay with CAFs and Cancer Cells
245
streptomycin and 1% of MEM Nonessential Amino Acid Solution (100Â).
3. Trypsin EDTA solution 1Â.
4. Phosphate buffered saline sterile.
2.2 Hanging-Drop
Method for Spheroid
Self-Assembly
1. Sterile syringe filter with a 0.22 μm pore size hydrophilic Polyethersulfone (PES) membrane, 33 mm diameter.
2. 1.2% Methylcellulose solution: Weight 0.6 g Methylcellulose
and transfer into a 50 mL tube with a magnetic stirring bar.
Make up to 50 mL with serum-free DMEM high glucose. Stir
until fully dissolved (it might take several minutes). Filter with a
0.22 μm filter, aliquot and store at 4
C (see Note 2).
3. CAFs culture medium (see Note 3), warmed at 37
C
before use.
4. 0.5% Trypsin EDTA, warmed at 37
C before use.
5. PBS buffer, sterile.
6. Cell labeling: In order to visualize the different population of
cell in the invasion assay, several strategies of cell labeling by
fluorescent dyes can be used. In this protocol we use CellTracker™ Green CMFDA Dye and Deep Red Dye staining
solution (Invitrogen) to stain the CAFs and cancer cells respectively (see Note 4).
7. Sterile Petri dishes (100 mm).
8. 1.5 mL sterile centrifuge tubes.
9. Hemocytometer chamber for cell counting.
10. Cell mixture: see Table 1.
2.3 Embedding
Spheroids into Matrix
1. 5ÂCol solution (see Note 5): Weigh 2.5 g of DMEM powder,
high glucose, pyruvate, and 1 g Sodium Bicarbonate
(NaHCO 3 ) transfer into a 50 mL tube. Add 5 mL of 1 M
Hepes pH 7.5 and make up to 50 mL with milli-Q water. Stir
until the powder is completely dissolved. Filter with a 0.22 μm
filter and store at 4
C.
2. Rat tail collagen type I. Store at 4
C. The stock concentration
ranges between 8 and 11 mg/mL (see Note 6).
3. Matrigel matrix (see Note 7). Make aliquots and store at
À20
C. Avoid multiple freeze-thaws. Thaw overnight at
4
C in ice one day before use.
4. Fetal bovine serum (FBS), filtered with a 0.22 μm filter. Keep at
4
C until use.
3D Invasion Assay with CAFs and Cancer Cells
245