TITLE: Wound healing migration/invasion assay in 96-well format
AUTHORS: Douglas Adamoski, Sandra Martha Gomes Dias

[STEPS]
SECTION: Collagen from rat tail tendon purification
1. This first part of the protocol is aimed to prepare the Collagen from rat tail tendon. If your lab already has collagen suitable for 3D matrix polymerization, you may skip this step.
 


 

SECTION: Collagen from rat tail tendon purification
2. Remove 10-15 rat tails from -80 °C  and left at 4 °C for thawing. 1440 min 
SECTION: Collagen from rat tail tendon purification
3. Move tails from 4 °C  to Room temperature . 30 min 
SECTION: Collagen from rat tail tendon purification
4. Wash tails in ultrapure water (in a large beaker) and transfer to a new beaker with ultrapure water.
SECTION: Collagen from rat tail tendon purification
5. Remove one tail and dry it with a paper towel.
SECTION: Collagen from rat tail tendon purification
6. Hold tail 5 mm before its thinner end using tweezers. Hold it and, using hands, twist the tail around the tweezer until skin break. Pull the tweezer with the collagen fibers.

SECTION: Collagen from rat tail tendon purification
7. Cut the fibers and immerse them in PBS 1X (room temperature). Repeat several times until the tail completely processed (or impossible to handle). Transfer to new PBS for 5 min . 

SECTION: Collagen from rat tail tendon purification
8. After collecting all fibers, transfer them to a new beaker with 500 mL  of PBS and wait for 5 min 

SECTION: Collagen from rat tail tendon purification
9. Transfer again to 500 mL  of pure acetone (inside chemical hood) and left stay for   . The fibers will lose their flexibility. 

 

SECTION: Collagen from rat tail tendon purification
10. Transfer to 500 mL  70 % volume Isopropanol (350 mL of isopropanol and 150mL of ultrapure water) and left stay for 5 min 

 

SECTION: Collagen from rat tail tendon purification
11. Collect fibers in a large beaker with a magnetic bar and add 500 mL  of acetic acid 20 millimolar (mM) (574 µL glacial acid acetic in 500 mL of ultrapure water).

SECTION: Collagen from rat tail tendon purification
12. Stir for at least 2880 min  at 4 °C. If the solution becomes too dense during this period, add  the needed amount of 20 millimolar (mM) acetic acid solution.

SECTION: Collagen from rat tail tendon purification
13. During the incubation, prepare small ice pieces with ultrapure water and store a household blender  and centrifuge flasks at 4 °C 

SECTION: Collagen from rat tail tendon purification
14. The solution should be viscous. Transfer the ultrapure ice to cup together with the collagen solution to the blender and pulse it multiple times (to avoid overheating). Just add a small amount of ice, enough to blend and keep the solution cold during the process. The solution will acquire a pale white color. Perform the steps at 4 °C 

SECTION: Collagen from rat tail tendon purification
15. Transfer to centrifuge flasks and balance them.  Immediately wash the blender to avoid protein binding to the plastic.

SECTION: Collagen from rat tail tendon purification
17. Transfer (without disturbing the pellet) to plastic or metallic containers to form ice. The final ice layer should be thinner than 5 mm.
SECTION: Collagen from rat tail tendon purification
18. Store at -20 °C  until completely frozen  2880 min .

SECTION: Collagen from rat tail tendon purification
16. Centrifuge for 10000 x g, 60 min, 4 °C 

SECTION: Collagen from rat tail tendon purification
19. Remove the frozen collagen, crush it to smaller pieces and transfer to the freeze-dryer flask. Perform the lyophilization 5760 min . Thaw the condenser coil during the process, if needed.

SECTION: Collagen from rat tail tendon purification
20. Prepare 5 L 20 millimolar (mM) of acetic acid and store at 4°C, together with a simple household hand mixer.

SECTION: Collagen from rat tail tendon purification
21. After finishing the lyophilization, remove collagen sponge, transfer to ziplock bags, and store at -80 °C .

SECTION: Collagen from rat tail tendon purification
22. In order to dissolve it, weight the desired amount (suggestion: 4 mg/mL ) and transfer this amount of collagen sponge to a beaker flask. Add enough acetic acid 20 millimolar (mM)  to achieve the desired concentration.

SECTION: Collagen from rat tail tendon purification
23. Dissolve the collagen using the hand blender. Use the following video as reference.

 

SECTION: Collagen from rat tail tendon purification
24. Assembly dialysis with 20 millimolar (mM) acetic acid (1:10, 4 °C ) for 120 min .

SECTION: Collagen from rat tail tendon purification
25. Change the dialysis buffer and left it   at 4 °C  with the magnetic bar.

SECTION: Collagen from rat tail tendon purification
26. Transfer the solubilized and dialyzed collagen to a sterile borosilicate glass flask with lid. Carefully add chloroform (10% of the total volume of collagen) at the bottom of the flask. Avoid mixing the chloroform with the collagen. Incubate for 1440 min  at 4 °C 
 



SECTION: Collagen from rat tail tendon purification
27. Carefully remove the chloroform inside biological safety cabinet using aseptic technique and sterile tools. Left it open inside the biological safety cabinet on on ice, for 60 min in order to remove chlorine gas.

SECTION: Collagen from rat tail tendon purification
28. Test for sterility and store at 4 °C  until further use

SECTION: Plate coating with Collagen I
29. Using pre-cooled tips (4 °C ) and in laminar flow hood, prepare Collagen COATING solution (see table below, solution enough for one 96-well plate). First, add the Acetic Acid 20 Mass Percent , then – while the stock solution is on ice  – collect Collagen I with a positive-displacement pipette.



 
     Collagen COATING solution        Reagent      Stock      Use      Volume        Collagen I, from rat tail      4      mg/mL      0,3      mg/mL      450,0      μL        Acid Acetic      20      mmol/L      --      5,6      mL               Total      6      mL        Prepare fresh and on sterile conditions   with pre-cold tips. Keep solutions on ice.   

 
 
SECTION: Plate coating with Collagen I
30. Briefly vortex and spin the conical tube. Dispense 50 µL  of Collagen COATING solution on each well from 96-well plate. Gently rock the plate to even coating of each well.

SECTION: Plate coating with Collagen I
31. Place the plate in a 37 °C incubator, 5 % volume , 60 min 

SECTION: Plate coating with Collagen I
32. Remove theplate from the incubator and aspirate the solution.
SECTION: Plate coating with Collagen I
33. Wash twice with phosphate-buffered saline.
SECTION: Plate coating with Collagen I
34. Dry out the plate in the hood, seal, and store at 4 °C 4 or immediately use.

SECTION: Wound healing assay
35. Tripsinize cell lines intended to use in the assay. Count using the appropriate device (eg. Neubauer chamber). Prepare a final solution with an adequate number of cells to coated plate in 100 µL . Check the suggested amount in the table below.

 


SECTION: Wound healing assay
36. Allow the cell plate to sit at room temperature for 5 min  to allow cells to evenly disperse across the bottom of the plate. This may require more or less time for each cell type.
SECTION: Wound healing assay
37. Place the plate in a 37 °C  incubator,  5% CO2, 720 min  -  1440 min 

SECTION: Wound healing assay
38. Remove medium from cells and wash them with serum-free medium.
SECTION: Wound healing assay
39. Add  150 µL of Serum-free medium supplemented with 0,1% HI-BSA

 


 

 
SECTION: Wound healing assay
40. Place the plate in a 37 °C  incubator, 5% CO2, 720 min  - 1440 min 

SECTION: Wound healing assay
41. Remove the plate from the incubator and perform scratch in all wells. 

Suggestion: Accessory 4 or 5 (both with attached 200 µL pipette tips) from Gilson's Aspiration Station Kit could be used to perform even and reproducible scratches. 

Check on the microscope if you were successful in the process.


 



SECTION: Wound healing assay
42. Remove the medium with aid of a multichannel pipette. Look to check if all wells have an integral scratch
SECTION: Wound healing assay
43. Wash the plate once with serum-free medium. 
SECTION: Wound healing assay
44. Left the wells with 100 μL of serum-free medium.
SECTION: Wound healing assay
45. Put the plate over ice-covered with aluminum foil for 5 minutes.
SECTION: Wound healing assay
46. Remove medium from wells.
SECTION: Wound healing assay
47. Overlay each well with 50 μL of Collagen INVADING solution. Gently rock the plate to even coating of each well.


 

 

SECTION: Wound healing assay
48. Return cells to the incubator for 30 min 

SECTION: Wound healing assay
49. Overlay the 3D matrices with 100 μL of medium with 10% FBS. The medium should be pre-warmed and pre-equilibrated in the incubator.


 

SECTION: Wound healing assay
44. Overlay cells with 200 µL of medium supplemented with 10% FBS.
 

SECTION: Wound healing assay
45. Return cells to incubator for 30 minutes.
SECTION: Capture and evaluation
46. Immediately image plate 0h. In Operetta system (Perkin-Elmer), use 10X Long WD objective, Brightfield mode, and set 15% overlap in “Well” options. Select 9 fields for each well in square-shape. Incubate with 5% CO2 at 37°C during the imaging.
 

SECTION: Capture and evaluation
47. Perform readings at every hour for 1440 min .

SECTION: Capture and evaluation
48. After transfer to Columbus, export images from Columbus (Using “Single Plane Tiff” option) to your computer.
SECTION: Capture and evaluation
49. Remove all accessory files and left each folder just with images.
SECTION: Capture and evaluation
50. Run ImageJ script “Scratch_and_Wound_1-Sorter_n_Tiler_v0.3_overlap_mult.ijm”  

 

SECTION: Capture and evaluation
51. Rename the “Tiles” folder to “TimepointBegin” (if the folder is relative to 0H) or “TimepointEnd” (if the folder is from any other timepoint).
SECTION: Capture and evaluation
52. Create an folder for each timepoint and put inside just “TimepointEnd” (the actual timepoin) and “TimepointBegin” (0H timepoint) folders. “TimepointBegin” will be the same for all “TimepointEnd”.
SECTION: Capture and evaluation
53. Run ImageJ script “Scratch_and_Wound_2-Measure_v0.2.1.ijm”.


 

50. Immediately image plate 0h. In Operetta system (Perkin-Elmer), use 10X Long WD objective, Brightfield mode, and set 15% overlap in “Well” options. Select 9 fields for each well in square-shape. Incubate with 5% CO2 at 37°C during the imaging.
 

51. Perform readings at every hour for 1440 min .

52. After transfer to Columbus, export images from Columbus (Using “Single Plane Tiff” option) to your computer.
53. Remove all accessory files and left each folder just with images.
54. Run ImageJ script “Scratch_and_Wound_1-Sorter_n_Tiler_v0.3_overlap_mult.ijm”  

 

55. Rename the “Tiles” folder to “TimepointBegin” (if the folder is relative to 0H) or “TimepointEnd” (if the folder is from any other timepoint).
56. Create an folder for each timepoint and put inside just “TimepointEnd” (the actual timepoin) and “TimepointBegin” (0H timepoint) folders. “TimepointBegin” will be the same for all “TimepointEnd”.
57. Run ImageJ script “Scratch_and_Wound_2-Measure_v0.2.1.ijm”.