Collagen Degradation Assay

Materials
  1. Cells of ~ 80% confluency
  2. Reguler culture medium: DMEM+10%FBS+1%PS
  3. DMEM with 0.1% BSA, filtered sterile at 0.22um
  4. Trypsin-EDTA solution
  5. PBS
  6. 0.6% Triton X-100 solution
  7. Stain solution
  8. Destain solution
  9. 12-well Collagen coated plate
  10. Pipettes and tips
  11. Disposable Pasteur pipet (for aspiration)
  12. Sterile 50ml centrifuge tubes
  13. Laminar flow hood
  14. Centrifuge with 50ml tube adapters
  15. BioIncubator (37oC and 5% CO2 )
  16. Shaker
  17. Digital camera and graphic application program
Procedure
  1. Remove the medium in cell culture flask by aspiration.
  2. Wash the monolayer cells with sterile PBS, Aspirate liquid completely.
  3. To T75 flask, add 3.5ml Trypsin/EDTA solution. Shake gently to thoroughly wet all the cells.
  4. Put the flask in incubator (37oC) for 3-10 min until the cells detach from the flask.
  5. Using about 7ml culture medium rinse the cells to a corner in the flask and pipet up and down several time to break up cell clumps, then transfer to a 50ml centrifuge tube.
  6. Take 10-20 ul cell suspension to count the cell density with hemacytometer.
  7. Calculate and take the volume for certain amount of cells (50k) into a 50ml centrifuge tube, fill up with serum-free medium for every sample to the same volume (10ml).
  8. spin at 200 xg for 5min.
  9. Remove the supernatant by aspiration.
  10. Resuspend the cell pellet in 25ul DMEM containing 0.1% BSA and drop onto the center of the collagen coated plate.

    After aspiration, there mostlikely still some medium left if you leave the tube standing for a couple of minutes. In my case there is usually 10ul medium left from 10ml, so I add 15ul DMEM+BSA and take 25ul cell suspension onto the plate. However, you have to practise yourself to determine how much to be added.

  11. Cover the lid and carefully transfer to incubator. Incubate for 2 hours to let the cells settle down.
  12. Carefully add 2ml DMEM containing 0.1% BSA to each well and incubate for 4 days with a medium change in between.
  13. Wash and trypsinize the cells with 0.5 ml Trypsin-EDTA at 37oC for 20 min.
  14. Add 0.5 ml 0.6% triton X-100 solution and mix. Incubate at room temperature for 20 min to completely remove the cell residues.
  15. Remove the liquid. Add 1 ml/well stain solution and shake at room temperature for 1 hour.
  16. Destain for 30-60 min.
  17. Dry and take pictures.
 
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