3D Gel Invasion Assay of Gastric Cancer Cells with Fibroblasts

Materials and Reagents

1. Collagen-coated dish (Sanyo, IWAKI, catalog number: 4010-010 )
   
2. Transparent PET membrane 24 well 3.0 μm pore size (Corning, Falcon^®, catalog number: 353096 )
   
3. 24 well plate for use with cell culture inserts (Corning, Falcon^®, catalog number: 353504 )
   
4. Razor blades (Esbjerg, Feather, catalog number: FA-10 )
   
5. Microslide glass (Matsunami Glass Ind, catalog number: TF0215M )
   
6. Micro cover glass, 24 x 24 mm (Thickness NO.1: 0.12-0.17 mm) (Matsunami Glass Ind)
   
7. Gastric cancer cells (44As3) (Yanagihara K et al., 2005)
   Note: It’s established from gastric cancer patient.
   
8. Fibroblasts (CAF) (Fuyuhiro Y et al., 2011)
   Note: It’s isolated from surgical materials of gastric cancer patients.
   
9. Type I-collagen (Nitta Gelatin Inc., Cellmatrix Type I-P)
   
10. Matrigel matrix (Corning, catalog number: 356234 )
    
11. 3, 3’-dioctadecycloxacarbo-cyanine perchlorate (DiO) (Thermo Fisher Scientific, Molecular Probes™, catalog number: D-275 )
    
12. 1, 1’-dioctadecyl-3, 3, 3’, 3’-tetramethyllindo-carbocyanine perchlorate (DiI) (Thermo Fisher Scientific, Molecular Probes™, catalog number: D-282 )
    
13. Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T3924 )
    
14. Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P4333 )
    
15. Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, catalog number: D6046 )
    
16. RPMI-1640 medium (Sigma-Aldrich, catalog number: R8758 )
    
17. 10x Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, catalog number: D2429 )
    
18. 10x RPMI-1640 medium (Sigma-Aldrich, catalog number: R1145-500ML )
    
19. Fetal bovine serum (heat inactivated) (Sigma life science, catalog number: 172012-500ML , batch: S13C490)
    
20. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417 )
    
21. 4% Paraformaldehyde phosphate buffer (Wako Pure Chemical Industries, catalog number: 163-20145 )
    
22. Polyvinyl alcohol mounting medium (Sigma-Aldrich, Fluka, catalog number: 10981 )
    
23. Instant glue (Krazy Glue, catalog number: KG585 )
    
24. NaHCO₃ (Sigma-Aldrich, catalog number: S6014-500G )
    
25. NaOH (Sigma-Aldrich, catalog number: S5881-500G )
    
26. HEPES (Sigma-Aldrich, catalog number: H7006-25G )
    
27. Gel (0.2 mg/ml type I-collagen and 2.5 mg/ml matrigel matrix) (see Recipes)
    
28. Reconstitution buffer (see Recipes)
    

Equipment

1. Tweezers (Electron Microscopy Sciences, Dumont, model: No.5 )
   
2. 37 °C, 5% CO₂ incubator (LabX, Sanyo, model: MCO-19AIC )
   
3. Dissecting microscope (OLYMPUS CORPORATION, model: SZ61 )
   
4. Light source (OLYMPUS CORPORATION, KL1600LED )
   
5. Vibratome (DOSAKA EM, model: LinearSlicer PRO7 )
   
6. Confocal laser scanning microscope (ZEISS, model: LSM 780 )
   

Procedure