1. 6-well Transwell plate
2. BD collagen sollution (cat# 354236) (If use dried collagen, refer to method for the preparation of collagen solution) (4oC)
3. 10X DMEM, sterile (4oC)
4. 1N sterile NaOH
5. autoclaved MilliQ H2O
6. 1mg/ml INT solution in PBS
7. 50ml Falcon centrifuge Tubes
8. Sereological pipets (sterile)
9. Pipetters and tips
-Keep all reagents and operation sterile-
10. Larminar flow hood
11. Centrifugate (optional)
12. Ice

Procedure

1. Place collage, 10X DMEM, 1N NaOH and sterile H₂0 on ice.
2. To a 50ml centrifuge tube, add H₂O, 10X DMEM, 1N NaOH and collagen sollution as in table 1 to make about 2ml/well times the number of wells needed. Mix carefully by pipetting up and down and avoid producing bubbles.

table 1. Calculation of collagen gel components

1. Add 0.9ml/well for 6-transwell plate. spread evenly by gently shaking. incubate at 37^oC for at least 10 min to form a collagen pad. Keep the unused collagen solution on ice.
2. trypsinize cells by standard precedure. Count cells and allocate at 50K/well into fresh sterile tubes, pellet to remove trypsin and EDTA.
3. Resuspend cells in collagen solution at 50ul/well and put onto the center of the collagen pad just made.
4. Incubate at 37^oC for 30min.
5. Cover the cell/collagen plug with 0.9ml collgen solution and incubate at 37^oC for 30min.
6. Add 2ml culture medium onto the surface of the gel and fill the lower camb with 3 ml culture media.
7. Incubate at regular condition and change media periodically as needed (usually every 2-3 days).
8. At the 6th day replace the media with INT solution and incubate overnight.
9. Observe cell growth and take pictures.